Device and method for detection of analytes

ABSTRACT

The present invention provides assays and devices for detection of substances in liquid samples. The assays and devices utilize passive diffusion between a porous material and a porous membrane containing a specific binding pair member to enable detection of the substance of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/172,474, filed Jun. 29, 2011, now U.S. Pat. No. 8,541,242,which is a divisional application of U.S. patent application Ser. No.11/720,142, filed May 24, 2007, now U.S. Pat. No. 8,343,726, whichclaims priority to PCT/US05/042902, filed Nov. 23, 2005, which claimspriority to U.S. provisional application 60/630,152, filed Nov. 24,2004, each of which is hereby incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to devices and methods for detectingsubstances that are present in liquids. In particular, the inventionrelates to devices and methods for detecting small molecules, such aschemicals or biological products, that are present in liquid samplesderived from body tissues or the environment.

2. Description of Related Art

There are various types of devices and methods available in the art fordetecting substances in samples. A large segment of the field utilizesmembrane-bound molecules that specifically bind to the substance ofinterest or to a molecule that is bound to the substance of interest.The two main types of devices and methods are generally referred to aslateral flow and flow through. These tests are generally relativelyrapid (less than 1 hour to detect a substance) and sensitive (ng/mlrange).

In a flow through device, a sample is pulled through a membrane bycapillary action and the substance (analyte, antigen, etc.) is retainedon the membrane by binding to a specific antibody, receptor, peptide,etc. The binding is detected by binding of a second antibody or othermolecule that is coupled to either an enzyme (e.g., horseradishperoxidase), a colloidal particle (e.g., gold sol), or various otherlabels and particles (e.g., fluorescent labels, paramagnetic beads). Thebinding occurs very rapidly as the sample is pulled through the membraneand the membrane is then washed (buffer is pulled through the membrane)and the detection reagent is added. The result is a detectable signal,such as a spot of color, a line, a plus sign, etc.

In a lateral flow device, the sample wicks across a thin membrane bycapillary action and flows through a line of a reagent, such as anantibody or other binding component (binding peptides, receptors, etc.).In certain versions, the analyte has already been bound by an antibodywith a colored particle attached (e.g., gold sol, blue dextran bead,etc). This complex of antigen and antibody-gold is bound by the reagentline and a colored line appears. There is no washing involved and noliquid reagents are used, except that the sample may be diluted in abuffered solution before it is placed onto the conjugate pad whichcontains the antibody-gold sol as a dried reagent.

In other versions of the lateral flow device, the sample is usuallymixed with a buffer containing an antibody-enzyme conjugate. This isplaced onto the membrane, and wicking occurs by lateral flow along thethin membrane. A line is not immediately visible because a reagent mustbe added. Usually this is a colorless chemical that is converted into aninsoluble colored precipitate by the enzyme (e.g., horseradishperoxidase, etc.). This version must be washed to leach away the unboundenzyme, so there is an absorbent pad at each end of the membrane and themembrane is typically more porous than that used for gold-sol lateralflow (this facilitates washing).

Although the devices and methods currently available for detectingsubstances in liquid samples are suitable and effective for detectingmost substances of interest, there is a need for new devices and methodshaving improved speed, sensitivity, and ease of use.

BRIEF SUMMARY OF THE INVENTION

The present invention addresses needs in the art by providing a deviceand method for rapidly detecting substances in liquid samples, which aresensitive and easy to use. The present device and method enableclinicians to rapidly detect organisms, biological products such astoxins or other biological materials such as proteins, nucleic acids(DNA, RNA), and polysaccharides, and drugs or other man-made chemicals,in tissue samples. By tissue samples, it is meant any composition thatcontains biological material originating from one or more animal cellsor tissues (including the blood system), including, but not limited to,tissues (e.g., whole blood or fractions thereof, tumor tissue, urine,excrement or excretion products, such as feces, diarrhea) of humans andanimals (e.g., veterinarian samples from farm animals or companionanimals, meat intended for human consumption, such as hamburger, steak,bacon, eggs, prepared food). It thus includes liquid or semi-liquidsamples of biological materials that do not need dilution prior to usein the method or with the device of the invention. It likewise permitsthe detection of biological or chemical substances in environmentalsamples, including surface soil, subsurface soil, rock, and water, andsurface water. In addition, it can be used to detect airborne substanceswhere such substances can be captured and dissolved in a liquid. Forexample, aerosols can be solubilized or otherwise combined with a liquidto create a liquid composition, which can be used as a sample fordetection of a substance of interest.

In general, the method of the invention uses diffusion of a substancethrough a membrane to permit detection, either directly or indirectly,of that substance by a specific binding pair member. Unlike detectionmethods in current use, which rely on passing, in a unidirectionalmanner, a substance over or through a membrane containing a specificbinding pair member for the substance, the present methods do not relyon such unidirectional passing of substance over or through a membrane.Rather, the present methods rely on simple diffusion of a substancethrough, around, over, across, and about a membrane to detect thesubstance, with no uniform directionality of movement with respect tothe membrane being necessary. Surprisingly, it has been found thatsimple diffusion through, around, over, across, and/or about a membranecontaining a specific binding pair member for a substance of interest issufficient for rapid and sensitive detection of the substance.

Accordingly, the method of the invention generally comprises providing aliquid containing, or suspected of containing, a substance of interest;applying the liquid to a porous material, such as a pad, in a sufficientamount to at least partially wet the porous material; contacting theporous material with a porous membrane comprising a specific bindingpair member that is capable of binding, either directly or indirectly,the substance of interest; and detecting the presence or absence of acomplex comprising the specific binding pair member and the substance ofinterest, where the presence of such a complex indicates the presence ofthe substance in the liquid.

Broadly speaking, the device of the invention comprises anyconfiguration of components that permit practice of the method of theinvention. More specifically, the device of the invention comprises anyconfiguration of components that permit a liquid sample containing, orsuspected of containing, a substance of interest to be retained in apre-defined area or region of the device, where the area or regioncomprises a porous membrane comprising a specific binding pair memberthat is specific, either directly or indirectly, for the substance.Within this area, the sample can diffuse across, through, etc. themembrane.

In its most basic form, the device of the invention comprises (a) areceptacle comprising a porous material or pad that is capable ofabsorbing and transmitting a liquid, and (b) a porous membrane thatcomprises a specific binding pair member that is specific for asubstance to be detected. The receptacle and porous membrane are eachshaped to permit the porous membrane to be in direct contact with theporous material over at least a portion of the porous membrane thatcomprises the specific binding pair member. In embodiments, the pad andmembrane are in direct contact with each other over at least a portionof the porous membrane that comprises the specific binding pair member.The device can comprise a container containing the receptacle. Thedevice can comprise a holder for the porous membrane. In embodiments,the device comprises the container and holder in contact with eachother, the contact between the two elements causing the porous membraneand the porous material to be in direct contact with each other over atleast a portion of the porous membrane that comprises the specificbinding pair member. In addition, the device can comprise a sampleapplication pad and a wash solution receiving pad.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying Figures, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the written description serve to explaincertain principles of the invention. The Figures provide details ofcertain embodiments of the invention in order to help better explainvarious features of the depicted embodiments. Because the Figures depictexemplary embodiments of the invention only, they are not to beconstrued as limiting the scope of the invention to the particulardetails depicted in them.

FIG. 1 is a perspective view of a basic configuration of the device ofthe invention, comprising a porous membrane and a receptacle.

FIG. 2 is a perspective view of a configuration of the device depictedin FIG. 1, in which the porous membrane is in direct contact with theporous material.

FIG. 3 is a cross-section side view of a configuration of a device ofthe invention, comprising a container and holder directly connected toeach other by way of a flexible hinge.

FIG. 4A is a cross-section side view of a configuration of a device ofthe invention in which a wash solution receiving pad is connected to asample receiving and reaction pad.

FIG. 4B is a cross-section side view of an alternative configuration ofthe device depicted in FIG. 4A, in which the wash solution receiving padis located below the sample receiving and reaction pad.

FIG. 4C is a cross-section side view of a configuration of the device ofthe invention in which a removable liquid impermeable barrier is locatedbetween a sample receiving and reaction pad and a wash solutionreceiving pad.

FIG. 5 depicts a side view of a configuration of the device of theinvention in which the sample receiving and reaction pad is extendedbeyond the reaction area, to form a unit comprising a separate samplereceiving area and a reaction area.

FIG. 6 depicts a cross-section side view of the device depicted in FIG.5 contained within a container that comprises a sample application portand a viewing window.

FIG. 7 depicts a cross-section side view of a configuration of thedevice of the invention comprising a sample application port and aviewing window.

FIG. 8 depicts a cross-section side view of a configuration of thedevice of the invention in which the holder for the porous membraneexerts pressure on the reaction pad at the site of the membrane to causecompression of the pad at this area.

FIG. 9 depicts a cross-section side view of a configuration of thedevice of the invention in which the container exerts pressure on thereaction pad from underneath at the site of the membrane to causecompression of the pad at this area.

FIG. 10 depicts a top view of a configuration of the device of theinvention, showing a sample application port and viewing window.

FIG. 11 depicts a top view of an alternative configuration of the deviceof the invention, showing a sample application port and viewing window.

FIG. 12 depicts a top view of the device depicted in FIG. 11, with areascomprising specific binding pair members and/or control molecules on themembrane within the area defined by the viewing window.

FIG. 13 depicts a cross-section top view of a configuration of thedevice of the invention, showing a single sample application padbifurcating to two separate reaction pads and porous membranes, whichare each connected to two separate wash solution receiving pads.

FIG. 14A depicts a top view of a configuration of the device of theinvention in which the sample application pad extends beyond the areadefined by the container.

FIG. 14B depicts a cross-section side view of an embodiment of theconfiguration of the device of the invention depicted in FIG. 14A.

FIG. 14C depicts a cross-section side view of an embodiment of theconfiguration of the device of the invention depicted in FIG. 14A.

FIG. 15A is a side view of a configuration of the device of theinvention in which a “clam shell” container is used.

FIG. 15B is a top view of the bottom half of the device depicted in FIG.15A.

FIG. 15C is a cross-section from the side of the device depicted in FIG.15A, where the top half is positioned above, but not in contact, withthe bottom half, and where the hinge is removed to permit alignment ofthe top and bottom halves for descriptive purposes.

FIG. 15D is a cross-section from the side of the device depicted in FIG.15A, in which the top half and bottom half are joined by friction fit.

FIG. 16 depicts a cross-section side view of a configuration of thedevice of the invention in which the device comprises two sectionsconnected by a hinge.

FIG. 17 depicts a side cross-section of a configuration of the device ofthe invention in which the device comprises an impermeable barrier.

FIG. 18 depicts a side cross-section of a configuration of the device ofthe invention in which the device comprises an impermeable barrier.

FIG. 19 depicts a side cross-section of a configuration of the device ofthe invention in which the device comprises a hinge connecting the topand bottom halves. Panel A depicts the device in a closed position forsample application and binding. Panel B depicts the device in an openposition for reading reaction results.

FIG. 20 depicts a side cross-section of a configuration of the device ofthe invention in which the device comprises an application port at thebottom of the device and a wash solution receiving pad on the side.

FIG. 21A depicts a top view of a configuration of the device of theinvention, as it would look when a positive sample were detected.

FIG. 21B depicts a top view of a configuration of the device of theinvention, as it would look when a negative sample were detected.

FIG. 21C depicts a top view of a configuration of the device of theinvention, when the device and/or method failed.

FIG. 21D depicts a top view of a configuration of the device of theinvention, when the device and/or method failed.

FIG. 22 depicts a table illustrating an age distribution of patients ofthe method of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Before proceeding with a description of the invention and variousembodiments, certain terms used herein will be defined at this point.Other terms used herein are used in accordance with their normaldefinition in the art or are defined at some other point herein.

As used herein, a substance is anything, including, but not necessarilylimited to, simple, natural organic molecules such as sugars andshort-chain acids; complex biological molecules such as peptides,nucleic acids (e.g., DNA, RNA, PNA), and polysaccharides; and man-made(whether though manipulation of biological processes or through chemicalsyntheses) molecules such as drugs, industrial agents, pesticides, anddefoliants. Thus, a substance can be a drug, hormone (such as onepresent during pregnancy or ovulation), protein (including antibody),toxin, DNA (including single-stranded DNA), RNA (includingdouble-stranded RNA), virus or viral protein or nucleic acid, bacteriumor bacterial protein or nucleic acid, polysaccharide, pollutant, and thelike. It can thus be a bacterial or viral pathogen or a prokaryotic oreukaryotic parasite.

For example, the substance can be a living organism or virus, or anypart thereof, including, but not limited to, macromolecules. Thus, thesubstance can be a gram positive or gram negative procaryotic organism,such as a Eubacterium or Archaea. Non-limiting examples of bacterialorganisms include Clostridium species, such as C. difficile, C. tetani,C. botulinum, and C. perfringens; Escherichia coli; a Salmonellaspecies, such as Salmonella typhimurium, and Salmonella typhi; aBacillus species, such as Bacillus anthracis and B. cereus; aStaphylococcus species, such as S. aureus and S. epidermidis; aStreptococcus species, such as S. pyogenes, S. mutans, and S.pneumoniae; a Neisseria species, such as N. meningitidis and N.gonorrhoeae; a Haemophilus species, such as H. influenzae; a Bordetellaspecies, such as B. pertussis, B. parapertussis, and B. bronchiseptica,a Listeria species, such as L. monocytogenes; a Corynebacterium species,such as C. diphtheriae, and C. pseudotuberculosis; a Mycobacteriumspecies, such as M. tuberculosis, M. bovis, M. scrofulaceum, M.avium-intracellulare, and M. leprae; an Actinomycetes species; aKlebsiella species, such as K. pneumoniae; a Serratia species, such asS. marcescens; a Proteus species, such as P. mirabilis and P. vulgaris;a Shigella species, such as S. flexneri; a Vibrio species, such as V.cholerae; a Pseudomonas species, such as P. aeruginosa; a Yersiniaspecies, such as Y. pestis; a Francisella species, such as F.tularensis; a Brucella species, such as B. abortus, B. suis, and B.canis; a Treponema species, such as T. pallidum; a Borrelia species,such as B. burgdorferi; a Campylobacter species, such as C. jejuni andC. fetus; a Legionella species, such as L. pneumophila; a Rickettsiaespecies; a Chlamydia species, such as C. trachomatis and C. psittaci;and a Mycoplasma or Acholeplasma species.

Of course, the substance can be a virus or any part thereof.Non-limiting examples of viruses include immunodeficiency viruses, suchas human immunodeficiency viruses (e.g., HIV-1, HIV-2, HIV-O); hepatitisviruses, such as hepatitis C virus (HCV), and hepatitis B virus (HBV);papilloma viruses, such as human papilloma virus (HPV); and any othervirus associated with human or animal disease.

Because the substance can be any portion of a living or non-livingentity, the substance can be a protein or portion thereof associatedwith a neurodegenerative disease, such as Alzheimer's disease or atransmissible spongiform encephalopathy, such as a prion disease. Thus,the substance can be a prion protein or portion thereof.

Other non-limiting examples of substances include parasites or anyportion thereof. Thus, substances can be all or part of a Giardiaspecies, a Cryptosporidium species, or an Entamoeba species.

As used herein, a specific binding pair member is a substance thatspecifically binds, either directly or indirectly, to another substance.Thus, together, the specific binding pair member and the other substancecreate a pair of substances. Because the two substances specificallybind each other, both can be considered specific binding pair membersfor each other. However, for clarity purposes, one will be referred toas the specific binding pair member and the other as a substance towhich it binds. Examples of specific binding pair members include, butare not limited to, antibody-antigen pairs (including, but not limitedto, antibody-antibody pairs where one antibody specifically bindsanother antibody), enzyme-substrate pairs, complementary nucleic acidpairs, protein-nucleic acid pairs (such as DNA and DNA-binding proteinpairs, including, but not limited to, an operator sequence and atranscription factor), and protein-protein pairs (including, but notlimited to, subunits of a multi-subunit protein). Examples also includeartificial peptides that bind to a target, receptors, ligands,artificial antibodies (e.g., single chain antibodies, recombinantantibodies, antibodies containing just the antigen binding region,bacterially produced antibodies or antibody parts). Other exemplarybinding pair members include two or more nucleic acids that containregions of complementarity, either over a portion or the entirety of oneor more of the nucleic acids, and which can specifically bind orhybridize under appropriate conditions. The invention is directed todetecting substances in a sample. However, it should not be assumed thatthe substances is necessarily one of the specific binding pair members.Rather, a specific binding pair member can be a substance thatspecifically reacts with another specific binding pair member, and atthe same time binds to a substance of interest in the sample (e.g., anantibody that binds to a substance of interest and at the same time isspecifically bound by another antibody). As used herein, disclosure of aspecific binding pair member (e.g., an antibody) binding to a substanceof interest in a sample includes not only direct binding of the specificbinding pair member to the particular substance, but to another specificbinding pair member that is bound to the substance. For example, aspecific binding pair may be biotin and avidin or streptavidin.

Rapid test devices and methods currently used in the art work becausethey force a substance (e.g., an antigen) and a specific binding pairmember (e.g., an antibody) to come together, either by sucking thesample through a membrane coated with the antibody (flow through devicesand methods) or by pulling it along a membrane by capillary action(lateral flow devices and methods). The present invention does not relyon either of these principles. According to the present invention, aporous membrane comprising the specific binding pair member simply needsto contact a porous material (e.g., a pad) that contains the substance.Simple diffusion in and out of the porous material and membrane causesthe substance and specific binding pair member to come into contact.That the present invention provides a rapid and sensitive test forsubstances in samples is surprising because it is widely held thatsimple diffusion is not sufficient for detection of a substance, muchless detection with high sensitivity by way of binding to a specificbinding pair member associated with a solid support, such as a membrane.Indeed, the present invention provides exceptionally sensitive detectionof substances at times comparable to those used for lateral flow or flowthrough devices and methods. Because the sample does not have to beapplied directly to, and does not necessarily flow directly through, themembrane comprising the specific binding pair member, the test also canbe used on samples that clog the membranes of other rapid tests.

In a first aspect, the present invention provides a method of detectinga substance that is present in a liquid sample. The method of theinvention uses passive diffusion of a substance from a receptaclecomprising a porous material, such as a pad, to a porous membrane. Suchdiffusion permits detection, either directly or indirectly, of thesubstance by a specific binding pair member associated with the porousmembrane. Unlike common detection methods in current use, which rely onpassing, in a unidirectional manner, a substance across or through amembrane containing a specific binding pair member for the substance,the present methods do not rely on such unidirectional movement of thesubstance. Rather, the present methods rely on simple diffusion of asubstance through, around, over, across, and/or about a membrane todetect the substance, with no single directionality of movement withrespect to the membrane being necessary. Surprisingly, it has been foundthat simple diffusion through, around, over, across, and about amembrane containing a specific binding pair member for a substance ofinterest is sufficient for rapid and sensitive detection of thesubstance.

The method of the invention generally comprises providing a liquidcomprising or suspected of comprising a substance of interest; applyingthe liquid to a porous material in a sufficient amount to at leastpartially wet the porous material; contacting the porous material with aporous membrane comprising a specific binding pair member that iscapable of binding, either directly or indirectly, the substance ofinterest; maintaining the wetted porous material and the porous membranein contact for a sufficient amount of time for the porous membrane tobecome wetted at least in the area comprising the specific binding pairmember; and detecting the presence or absence of a complex comprisingthe specific binding pair member and the substance of interest, wherethe presence of such a complex indicates the presence of the substancein the liquid.

The liquid comprising or suspected of comprising a substance of interestcan be provided in any number of ways. For example, it can be providedin the form that it was isolated from its natural environment (e.g.,whole blood, urine, diarrheal feces, and stream, river, or lake watercan be used directly as isolated). Thus, it can be an undiluted sample.Alternatively, it can be provided in a form after having been treated toremove one or more components (e.g., the liquid portion of blood andfeces can be used after centrifugation, filtration, or precipitation ofsolid matter). In addition, where the original sample is solid orsubstantially solid, a liquid, such as water, may be added to the sampleto provide liquid characteristics. Other handling or manipulation of theliquid can be performed prior to or at the time of providing the liquid.Any handling or manipulation may be used as long as it does not renderthe sample incapable of use in the method of the invention or in thedevice of the invention.

The liquid can be any liquid, including, but not limited to, water orcompositions containing water, such as biological tissues, extracts ofbiological tissues, and biological excretions; organic solvents orcompositions containing organic solvents; and combinations of water andorganic solvents or combinations of aqueous and/or organic solventcompositions. For example, the liquid can be a biological fluid, such asblood or a portion of blood, urine, feces, saliva, sputum, mucous,semen, or homogenized tissue. It thus can be a homogenized sample ofhuman or animal tissue, such as homogenized meat (e.g., hamburger, lamb,pork, chicken, fish, egg). It also may be an extract of a solidspecimen, such as an aqueous extract of a fecal sample or of aconsumable meat sample. Where the tissue to be analyzed is not suitablefor liquification as isolated, water or another liquid may be added tothe tissue to provide suitable liquid characteristics. Because thepresent invention is suitable for detection of substances in liquidshaving a wide range of viscosities, the present method is suitable fordetecting substances present in liquid or semi-liquid compositions.

Where necessary, the amount or concentration of the substance to bedetected within the liquid, if present, can be adjusted to achievesatisfactory detection. Adjustment can be accomplished by dilution witha liquid that is compatible with the liquid sample and the components ofthe device of the invention, or can be accomplished by concentration ofthe substance within the liquid sample using any suitable concentrationtechnique, including, but not limited to, centrifugation, filtration,evaporation, affinity purification, or the like. In general, thesubstance to be detected is present in the sample in nanogram (ng) tomicrogram (ug) amounts.

Additional components may also be added to the liquid prior to, or atthe time of, applying of the liquid to the porous material. Anythingthat does not interfere substantially with the ability of the specificbinding pair member to specifically complex with the substance or aspecific binding pair member that binds to the substance may be added tothe liquid. In embodiments, a label that specifically interacts with thesubstance, if present, is added to the liquid prior to or at the time ofapplying the liquid to the porous material. For example, an antibodythat specifically binds to the substance of interest can be added to theliquid prior to applying the liquid to the porous material. The antibodymay be labeled with a moiety that can be detected, either directly orthrough the use of ancillary materials. Exemplary moieties include, butare not limited to, alkaline phosphatase, horseradish peroxidase,fluorescent compounds, paramagnetic beads, gold or other metals, latexbeads, avidin (streptavidin), and biotin. Thus, in embodiments, anantibody conjugate that specifically binds to the substance of interestis added to the liquid sample prior to or at the time of applying theliquid sample to the porous material/pad.

The liquid is applied to the porous material in a sufficient amount toat least partially wet the porous material. It is preferred that asufficient amount of liquid is applied to wet that portion of the porousmaterial that is in contact with the porous membrane at the region wherethe specific binding pair member is located. In preferred embodiments,the entire porous material or substantially the entire porous materialis wetted. In embodiments where the porous material extends beyond thearea covered by the porous membrane, it is preferred that a sufficientamount of liquid is applied to wet the porous membrane at least at theregion where the specific binding pair member is located.

Applying can be accomplished by any suitable technique, including, butnot limited to, dipping of the porous material into the liquid, pouringthe liquid onto the porous material, placing the porous material in thepath of a stream of liquid (e.g., dipping into a flowing river,inserting into a stream of urine), dropping the liquid onto the porousmaterial (e.g., with an eye dropper or pipette), and smearing asemi-liquid sample onto the porous material. Applying can beaccomplished by direct application to the porous material or to anotherporous material in contact with the porous material. Likewise, it can beaccomplished by applying to an area of the porous material that isphysically distant from another site, and permitting the liquid tomigrate through the porous material to the other site.

In practice of the invention, at least a portion of the liquid that isapplied should be present at a site directly in contact with a portionof the membrane comprising a specific binding pair member. Thus, theliquid can be applied to a portion of the porous material adjacent thearea where the porous membrane is in contact with the porous material oris intended to be in contact with the porous material (e.g., in thereaction pad area, which includes the detection area or site) or can beapplied at a site distal to the reaction pad area, and allowed tomigrate to that area. When applied at a distant site, due to theporosity of the material, the liquid will travel through the porousmaterial from the site of application to the site of detection of thepresence of the substance (i.e., to the site on the porous materialwhere the porous membrane is in contact).

Applying the liquid at a site away from the site of detection can permitfiltration of the liquid before detection of the substance. That is, theporous material can act not only to transport the liquid and itscomponents to the site of detection, but can also act to block or retardmigration of certain components present in the sample, thus effectivelyacting as a filtration system that permits substances of a certain sizeonly to migrate to the detection area. Numerous different porousmaterials are available, having various different pore sizes, and onemay select the appropriate material and pore size to effectively filterout unwanted components in the liquid. For example, when applying aliquid comprising feces, one may want to filter out large particles,such as undigested or partially digested food or bacteria. In such asituation, one may select a porous material that has a pore size thatblocks or significantly retards migration of these relatively largecomponents while permitting smaller components, such as bacterialproteins, nucleic acids, extracellular blood proteins, or the like, tomigrate through the material essentially unimpeded.

The method of the invention also comprises contacting the porousmaterial with a porous membrane comprising a specific binding pairmember that is capable of binding, either directly or indirectly, thesubstance of interest. Contacting of the porous material and the porousmembrane can occur prior to or after applying the liquid to the porousmaterial. Furthermore, it is not relevant whether the material or themembrane is caused to move in order to effect contact. Contactingencompasses physical movement of either or both the porous material andporous membrane to achieve contact.

Although not necessary, typically when the liquid is to be applied to aporous material at a site away from the site of detection of thepresence of the substance, the porous material and porous membrane arecontacted with each other prior to applying the liquid. On the otherhand, when the liquid is applied to the porous material at or very nearthe site where the porous material and porous membrane make contact, theliquid is typically applied before the membrane and material arecontacted.

It has been found that direct contact of the membrane at the site wherethe specific binding pair member is located with a porous materialthrough which a substance of interest can travel improves thesensitivity and speed of the method of the invention. Therefore, it ispreferred that the porous membrane and porous material are in contact atthis site, or at least over a portion of this site. Contact between theporous material and porous membrane should be continuous contact over atleast the portion of the membrane where the specific binding pair memberis located, or over a sufficient portion of the membrane at the sitewhere the specific binding pair member is located that a detectablesignal can be identified if the substance of interest is present in theliquid. That is, in embodiments, the area where the specific bindingpair member is bound to the porous membrane may exceed the area ofdirect contact with the porous material, but a sufficient amount ofcontact will be made such that the presence of the substance in theliquid can be detected.

Direct contact of the membrane and the material is preferred; however,one or more intervening porous, substantially hydrophilic materials maybe interposed between the porous material and the porous membrane. Insuch a situation, the intervening porous materials effectively act assecondary porous materials, and thus can be considered for the purposesof the invention, to be a porous material or pad. Thus, use of the termsporous material or pad encompasses multiple materials that provide thesame or essentially the same function.

In embodiments, contacting of the material and membrane comprisesexerting pressure on the membrane and material to ensure complete oressentially complete contact of the two over at least a portion of thearea where the specific binding pair member is located. While notnecessary, it has been found that, under certain circumstances, pressureat this area may improve the performance of the method. For example, itcan improve the sensitivity and reliability of the method. It can alsoimprove the wicking characteristics of the porous material, which canincrease the amount of sample in the reaction area. Pressure can furtherincrease the amount of contact between the porous material and theporous membrane. That is, in embodiments where the porous materialcomprises both a sample application site and a reaction site, it hasbeen found that compression of the porous material at the reaction sitecan improve the sensitivity of the device, and thus the assay or methodof the invention. While not desiring to be limited to any particulartheory of operation, it is believed that, in addition to improving thecontact between the membrane and the material, compression of thereaction area of the porous material improves migration of liquid intothe area of the material in contact with the membrane, and in particularthe portion of the membrane comprising the specific binding pair member,and impedes migration of liquid out of the area. In effect, thecompression causes the liquid to collect in the area of compression. Theimproved migration into, but not out of, the area causes an increase inthe amount of substance in the area (as compared to an uncompressedmaterial) and enhances diffusion of the substance (if present) into theporous membrane.

According to the method of the invention, the wetted porous material andthe porous membrane are maintained in contact for a sufficient amount oftime for the porous membrane to become wetted at least in a portion ofthe area comprising the specific binding pair member. Doing so permitsthe substance, if present in the sample, to diffuse through, over,around, and/or about the membrane and make contact with the specificbinding pair member associated with the membrane. Although the amount oftime provided will vary depending on the amount of substance in thesample, the porosity of the porous material and membrane, the amount ofspecific binding pair member associated with the membrane, thespecificity and strength of binding of the specific binding pair memberto the substance, the temperature, and other factors (all of which canbe selected by those of skill in the art without undue experimentationbased on times, concentrations, temperatures, etc. generally used in theart for rapid tests), typically, sufficient wetting of the membraneshould occur within one minute. In preferred embodiments, the membraneand material are maintained in contact for at least thirty seconds, suchas about or precisely thirty seconds, about or precisely one minute,about or precisely 2 minutes, about or precisely 3 minutes, about orprecisely 5 minutes, about or precisely 10 minutes, about or precisely15 minutes, about or precisely 20 minutes, about or precisely 25minutes, or about or precisely 30 minutes. As used herein, unlessotherwise noted, times, temperatures, and other numerical values recitedinclude a range about the stated number of 5% at either end of therecited number. Thus, recitation of “60 seconds” includes any amount oftime from 57 seconds to 63 seconds.

Maintaining the membrane and material in contact can be performed at anytemperature. However, it is preferred that temperatures below 100° C. beused, such as room temperature (20°-25° C.), 30° C., 37° C., 40° C., or50° C. Indeed, it has surprisingly been found that the present methodcan provide sensitivities greater than ELISA tests using the samespecific binding pair member and substance, while being performed atroom temperature rather than 37° C. (as is needed for an ELISA).

Likewise, any suitable concentration or amount of specific binding pairmember and substance can be used. General amounts of various specificbinding pair members (e.g., antibodies, enzymes, nucleic acids) to beused for membrane-bound detection of binding partners (e.g., antigens,enzyme substrates, nucleic acids or nucleic acid binding proteins) areknown in the art. For example, when the specific binding pair member isan antibody, it can be present on the membrane at an amount of about 0.5ng to about 1000 ug and over an area from about 0.5 square mm to about100 square mm or more. Amounts to be bound to the membrane can beselected based on the amount of substance to be detected, theamount/intensity of signal intrinsically produced by the selected labeland signal generation system, and the size of the area on which thespecific binding pair member is bound. These parameters may be selectedand adjusted by those of skill in the art based on well knowncharacteristics of each signal generation system.

In embodiments, applying the liquid is performed before contacting theporous material with the porous membrane. In other embodiments, applyingthe liquid is performed after contacting the porous material with theporous membrane. Thus, in embodiments, the porous material and porousmembrane are in contact with each other before the liquid is applied. Ingeneral, it is not important whether contact between the porous materialand porous membrane occurs before or after application of the sample.The time at which contact is made is typically selected in conjunctionwith the configuration of the device used for a particular assay, andthe ease of use of the device.

The method further comprises detecting the presence or absence of acomplex comprising the specific binding pair member and the substance ofinterest, where the presence of such a complex indicates the presence ofthe substance in the liquid. According to the invention, if a samplecontains a substance of interest, diffusion of the sample between theporous material (i.e., reaction pad) and porous membrane will permit thesubstance to come into contact with the specific binding pair member,which is bound to the membrane. The method of the invention detects thecomplex formed from the specific binding pair member and substance byany of a number of art-recognized detection schemes. For example, anantibody (other than the specific binding pair member bound to themembrane) that is specific for the substance can be exposed to thesubstance, either before the substance is exposed to the specificbinding pair member (e.g., before the sample is applied to the porousmaterial, while the sample is migrating through the porous material,etc.) or after sufficient time has been provided for the substance andspecific binding pair member to come into contact. In some situations,the antibody will be labeled with a detectable moiety, such as with alabel that can be directly detected (e.g., a metal sol, such ascolloidal gold; a dye sol; a colored particle, such as latex); aparamagnetic bead; and a fluorescent compound. It can also be labeledwith an indirect label, such as an enzyme that produces a detectablesignal when exposed to a substrate (e.g., horseradish peroxidase,alkaline phosphatase). In other situations, the antibody will serve as aspecific binding pair member for a label, such as by binding of thelabel to the Fc portion of the antibody. Labels can also includespecific binding pairs in which one or both members contain a detectablemoiety or substance that can generate a detectable moiety, such as anavidin (streptavidin)/biotin pair or any of its functional equivalents.

As alluded to in the previous paragraph, the label for the substance ofinterest may be provided as a component of the porous material. Forexample, it may be impregnated (as a dry substance or as a liquidsolution that is permitted to dry in the porous material) in thereaction zone, the sample application zone, or the sample migration zone(located between the sample application zone and reaction zone incertain embodiments). As the liquid sample migrates into and through theporous material, the label is dissolved in the liquid and migrates alongwith the liquid to the reaction site and, eventually, to the membrane.During the migration process or during the reaction process, the labelspecifically binds the substance (if present), ultimately resulting in amembrane-bound complex comprising the specific binding pair member, thesubstance of interest, and the label. In embodiments where a label isincluded in the porous material, selection of the pore size of thematerial will depend, at least in part, on the migration characteristicsof the label or the substance-label complex.

Detection of the substance can provide qualitative, semi-quantitative,or quantitative information on the substance in the sample. Qualitativedetection provides information that informs the practitioner of thepresence, but not necessarily the amount, of the substance in thesample. However, by placing a known amount of specific binding pairmember on the membrane, by knowing the amount of substance that thespecific binding pair member can bind, and by knowing the amount ofsubstance-label complex that must be present for detection, one canprovide a method of detection of a substance that is semi-quantitative.More specifically, by knowing these amounts, and by obtaining adetectable signal, the practitioner will know that the substance is notonly present in the sample, but that the substance was present in atleast an amount necessary to produce the detectable signal. If desired,quantitative measurement of sample amount can be obtained by comparingsignal strength of the test sample with a standard curve of signalstrengths derived from samples containing known amounts of substance.Various ways of designing semi-quantitative and quantitative assays areknown in the art, and any suitable one may be used within thisinvention.

Detection may also be the absence of a detectable signal, or thediminution of a signal that would otherwise be generated in the absenceof the substance or some other substance that is indicative of thepresence of the substance. Thus, the methods of the invention encompassall types of immunoassays (including both sandwich-type assays andcompetitive assays) and any other assays that rely on detecting orfailing to detect binding of at least one specific binding pair memberto a substance of interest.

The method of the invention can comprise numerous other steps,including, but not limited to, providing one or more control reactionsto determine whether one or more steps in the method have been performedsuccessfully, to determine whether one or more reagents is functioningas expected, or to determine if substances that interfere with theability of the method to generate reliable results are present in thesample. Substances that can be used as control reagents include, but arenot limited to, the substance to be detected or a structural analog, anantibody that specifically reacts with another antibody, or anythingelse that can specifically bind to the substance of interest or someother reagent used in the assay. Accordingly, the method of theinvention can include adding a known substance, including the substanceto be detected, to determine if one or more steps of the method areworking as designed. Such control reactions are well-known to those ofskill in the art, and their design and implementation need not bedetailed herein. A common control reaction will comprise providing asecond area, either on the porous membrane that specifically binds thelabeled substance or on a second porous membrane, to show not only thatthe label is present and functional, but to show that the label has hadsufficient time to contact any binding partner associated with themembrane. Of course, multiple lines may be provided in variousorientations, each providing repetitive or different information aboutvarious aspects of the method.

In addition, the method of the invention can detect one or moresubstances in the sample in addition to the substance of primaryinterest. The other substance(s) can be other substances that naturallyoccur in the sample being tested, or can be substances that areintentionally added to the sample to serve as positive controls, labels,competitors, and the like. Thus, the methods of the invention can detecttwo or more substances in a sample. When doing so, the multiplesubstances can be detected on the same porous membrane, or multiplemembranes may be provided, either on the same device or on two identicaldevices (with the exception of the identity of the substance(s) bound tothe membrane).

The method of the invention may comprise one or more washing steps.Although not limited to any particular method, washing is typically usedin embodiments where indirect labels are used to detect the substance.For example, when using a label that uses a substrate to generate asignal (e.g., horseradish peroxidase), the label will typically bepresent in the reaction mixture in excess over the substance. Inaddition, it is possible that the substance-label complex might bepresent in excess over the specific binding pair member bound to themembrane. In either such situation, the excess label, if permitted toremain in and around the membrane comprising the specific binding pairmember, would react with the label substrate to produce a signal, whichwould represent non-specific signal or background noise. To reduce thisbackground noise, the membrane can be washed with an appropriate volumeof an appropriate wash solution. The wash solution can be applied onceor more than once, depending on the amount used and the amount ofunbound label present. Likewise, other wash steps may be included atother points in the method. In embodiments, the wash step is used towash unbound conjugate from the membrane to improve detectionsensitivity. Those of skill in the art are well aware of advantages anddisadvantages of performing or not performing washing steps at variouspoints during specific binding reactions, and can thus select the typeand number of washing steps, as well as the washing solutions, to usefor each particular embodiment of the invention. Such selection can bemade without undue experimentation.

In a second aspect, the invention provides a device for practicing themethod of the invention. Broadly speaking, the device of the inventioncomprises any configuration of components that permit practice of themethod of the invention. More specifically, the device of the inventioncomprises any number and configuration of components or elements thatpermit a liquid sample containing, or suspected of containing, asubstance of interest to be retained in a pre-defined area or region ofthe device, where the area or region comprises a porous membranecomprising a specific binding pair member that is specific, eitherdirectly or indirectly, for the substance.

In its most basic form, the device of the invention comprises (a) areceptacle comprising a porous material that is capable of absorbing andtransmitting a liquid, and (b) a porous membrane that comprises aspecific binding pair member that is specific for a substance to bedetected (be it the substance in the sample or a substance that binds tothat substance), where the receptacle and porous membrane are eachshaped to permit the porous membrane to be in direct contact with theporous material over at least a portion of the porous membrane thatcomprises the specific binding pair member. The device can furthercomprise a container containing the receptacle or a portion of thereceptacle. The device can further comprise a holder for the porousmembrane. In embodiments, the device comprises the container and holderin contact with each other, the contact between the two elements causingthe porous membrane and the porous material to be in direct contact witheach other over at least a portion of the porous membrane that comprisesthe specific binding pair member.

The receptacle is a physical and functional unit of the device. Itprovides an area and volume for liquids containing or suspected ofcontaining a substance of interest to be retained. It also provides areservoir for liquid to diffuse into and out of the porous membranecomprising the specific binding pair member, which is referred to hereinas a reaction pad or zone. The receptacle can be of any shape and size,and fabricated of any suitable material. The receptacle comprises atleast one porous material, also referred to herein as a pad. However, itis to be noted that the porous material is not necessarily limited insize to the area defined by the receptacle. That is, a single porousmaterial or a combination of porous materials can be limited in size tothe receptacle area or can extend beyond the receptacle area to thesample application area and/or wash area, or any other area present in aparticular configuration of the device.

The porous material (also referred to herein as a “pad”) can befabricated from any material that has pores, holes, or spaces throughwhich one or more liquids can pass. It is thus any material that isabsorbent. Non-limiting examples of porous materials include, but arenot limited to, paper products, such as bibulous or filtration paper(e.g., Whatman® 3 mm paper, and Filtrona® products), synthetic polymericmaterials (e.g., nitrocellulose, nylon), plastics and plastic spheres(e.g., Porex® plastic beads; materials used in fabricating ballpointpens), such as those made from polypropylene, polyethylene,polyvinylidene fluoride, ethylene vinylacetate, acrylonitrile, andpolytetrafluoroethylene. Other non-limiting examples includenanoparticles/spheres/tubes.

The pore size of the material can be selected based on thecharacteristics desired. Numerous porosities are available for thevarious types of materials that the porous material can be made from.For example, if a sample to be applied contains particulate materials orsolids (e.g., feces, soil), a pores size that excludes or significantlyslows the migration of these particulate materials or solids can bechosen. Likewise, if the sample comprises blood, a pore size thatexcludes or significantly slows the migration of blood cells andplatelets can be chosen. Alternatively, if the sample does not containany substances that are undesirable at the detection site (e.g., asample that has been pre-purified to some extent), the pore size of thematerial may be selected without regard to filtration characteristics.In general, the pore size will range from about 0.05 micrometers toabout 0.5 micrometers.

The porous material may be fabricated from a single material or maycomprise multiple different porous materials. The differing individualmaterials may be configured in any suitable configuration, such aslayering one on top of another, abutting of two materials end-to-end, orany other configuration that permits a liquid to flow from one area ofthe material to another, such as from the site of application of aliquid to the site of detection on the porous membrane, when in contactwith the porous material. For example, the porous material may comprisea pore size and material of one type at the site of application of theliquid (the application zone), a second pore size and/or material at thesite of detection (the detection zone), and a third pore size and/ormaterial (which can be the same as the first) at a third area distal(with respect to the site of application of the liquid) to the detectionsite, the third site functioning as a wash solution receiving site (thewash receiving zone). The pore sizes and materials, and combinationsthereof, may be selected to suit individual needs based on the variouscharacteristics of the sample, the substance to be detected, theconfiguration of the device, or any other consideration. The porousmaterial may contain substances that are deemed useful in practicing theinvention, including, but not limited to, labels for the substance,activated charcoal, ion exchange resins, and surface active agents.Furthermore, the porous materials can be separated from each other byless porous, non-porous, or impermeable materials, such as hydrophobicmembranes. These membranes can be removed at some point during practiceof a method of the invention to permit flow of liquids from one or morematerials to one or more other materials. For example, the two porousmaterials may be separated by a non-porous material attached to apull-tab. Pulling of the tab removes the non-porous material and permitsflow of liquids, such as wash solution, into a porous material (e.g., awash pad).

The porous membrane is a membrane made of any suitable material thatpermits liquids and suspended substances of a pre-determined size toflow through it. Typically, the membrane is fabricated from materialsknown in the art to be suitable for detection of substances of interestby specific binding of a membrane-bound molecule to a substance.Examples include, nylon membranes, nitrocellulose membranes,polyvinylpyrolidone membranes, glass fibers, and the like.

The porous membrane comprises at least one specific binding pair member.The specific binding pair member is associated with the membrane in sucha way that it remains associated with the membrane under the conditionsof fabrication and use of the device. Typically, the specific bindingpair member is bound, by covalent, ionic, or hydrophobic bonds, to themembrane. The membrane may be treated prior to binding in order toenhance binding. Likewise, the membrane may be treated after binding toenhance binding or to reduce binding of other substances to sites on themembrane that are different than the site where the specific bindingpair member is bound. The specific binding pair member may be bound tothe membrane using any known technique. Furthermore, it may be bound tothe membrane in any shape, design, pattern, direction, etc. desired(e.g., a line, a cross, a dot, a circle) and in any size desired (e.g.,a dot of diameter of 0.1 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, etc.; a line1 mm thick and 5 mm long, 2 mm thick and 1 cm long, etc.). In preferredembodiments, it is in the shape of a line, which provides anadvantageous and convenient detection shape. In embodiments, the porousmembrane comprises two or more different areas containing a specificbinding pair member. In certain embodiments, two or more areas comprisethe same specific binding pair member. In other embodiments, each areacomprises a different specific binding pair member. The differentspecific binding pair members may be specific for the same substance (toprovide an internal reproducibility control), or may be specific fordifferent substances (e.g., one is specific for a substance of interest,while one or more others are specific for other substances of interestor for reagents used in the method of the invention).

Thus, the porous membrane may comprise other components in addition tothe specific binding pair member. For example, it may comprise amolecule to be used as a control for the performance of the method ofthe invention, such as an antibody that specifically binds the labelused to detect the substance of interest in the sample. It may alsocomprise a second specific binding pair member, the second specificbinding pair member being specific for a second substance of interest inthe sample (including a substance that is intentionally added to thesample to serve as a positive control). As discussed above, the specificbinding pair member may be an antigen or antibody in a binding pair, areceptor or ligand in a binding pair, or either component of a bindingpair. Thus, the present invention contemplates binding an antibody, anantigen, a receptor, a ligand, a single stranded nucleic acid, etc. tothe membrane in order to detect its binding partner.

In embodiments, the porous membrane is in contact with a holder. Theholder can hold the membrane in place such that the membrane stays incontact, over at least a portion of the membrane, with the reaction pad.It also can hold the membrane in a position such that it is capable ofbeing placed in contact with the reaction pad if so desired. Forexample, the holder can be a plastic ring, square, etc. that contactsthe membrane. Contact can either retain the membrane on the holder orsimply retain the membrane in contact with the reaction pad. The holdermay be a separate physical component of the device or it may befabricated as an integral portion of the device, for example as anintegral portion of the container of the device.

The receptacle may be located within a container. The container may befabricated of any suitable material, but is typically made of plastic.The container provides substantial structural strength and liquidimpermeability to the device of the invention, and may provide otherfunctions as well. In a basic configuration of the device of theinvention, the container contains the reaction pad, and comprises themembrane. The membrane can be provided in any suitable fashion thatpermits contact of the membrane with the reaction pad. For example, themembrane may be bound to a holder that is connected to the containerthrough a hinge. Alternatively, the membrane may be bound to a holderthat is integral with the container, where the container is fabricatedfrom two halves that fit together such that the membrane contacts thereaction pad. Other suitable configurations will be apparent to those ofskill in the art, and all such configurations are encompassed by theinvention.

The device of the invention can further comprise a sample applicationarea or zone (also referred to herein as the sample application pad)comprising a porous material. As described above, the sample may beapplied to the receptacle or reaction pad. However, in embodiments, thesample is added at a site distant to the receptacle and reaction pad. Insuch embodiments, the sample is added at the sample application pad,which comprises a porous material. The porous material of the sampleapplication pad may be the same material as that used in the reactionpad (i.e., it may physically be the same element or it may be twoseparate elements fabricated from the same material). Alternatively, itmay be a different material, where the two materials are placed incontact with each other so that liquid from the application pad may passinto the reaction pad.

The size of the sample application zone is not critical. However, it ispreferred that the sample application zone and the reaction zone,together, have sufficient absorbent ability to absorb the entire samplebeing applied. Furthermore, the sample application zone may comprise anopen area not containing the application pad, the area typically beingdefined by the edge of the application pad and the side of thecontainer. This open area may be designed to accept, either directly oras overflow, the sample being added, and may function to help filter andretain solids and large particles present in the sample.

The device may further comprise a wash solution receiving zone or area(also referred to herein as a wash solution receiving pad) comprising aporous material. The porous material of the wash solution receiving padmay be the same material as that used in the reaction pad and/or sampleapplication pad (i.e., it may physically be the same element as thereaction and sample application pads, or it may be a separate elementfabricated from the same material as one or both of the other pads).Alternatively, it may be a material that is different than either orboth of the reaction pad and the sample application pad. In such asituation, the wash solution receiving pad is placed in contact with thereaction pad, the application pad, or both, so that wash liquid from themembrane (flowing through the reaction pad) may pass into the washliquid pad.

In embodiments, a removable liquid impermeable or semi-permeable barrieris interposed between the wash solution receiving pad and one or moreother pads of the device. The barrier is present in embodiments toensure that no liquid from the reaction pad or application pad entersinto the wash solution receiving pad until the wash solution is added tothe device. While typically not necessary due to the liquid flow andretention characteristics of the reaction pad, the barrier may beincluded in the device for added assurance, to increase the amount ofliquid present in the reaction pad, or for any other reason. In otherembodiments, one or more liquid impermeable barriers are includedbetween one or more of the various pads present in the device.

The device may comprise a filtration pad or zone between the sampleapplication pad or zone and the reaction pad or zone. While the sampleapplication pad and reaction pad can provide suitable filtration of thesample, it may sometimes be desired to have additional filtration of thesample prior to exposure of the sample to the membrane. In suchsituations, a filtration pad may be provided. In embodiments, thefiltration pad is simply an extension of the reaction pad beyond thearea covered by the membrane. Alternatively, it can simply be anextension of the sample application pad that extends beyond the areawhere the sample is applied. In some embodiments, the sample applicationpad, filtration pad, and reaction pad are the same element, the various“pads” being designated (or referred to as “zones”) based on functionand location within the device rather than on physical characteristics.The primary function of the filtration pad is to block or retardmigration of certain substances to the reaction pad. Such filtration maybenefit the detection of the substance of interest by retarding coloredcompounds, retarding large particles that would cause background noise,and the like.

As can be seen from the above description, the reaction pad, sampleapplication pad, and filtration pad all may be fabricated from a porousmaterial (either the same material or different materials). Likewise,the wash solution receiving pad may be fabricated from a porousmaterial. The porous materials serve various functions, but generallyall serve to draw liquid through the material such that it passes toanother material. For example, the sample application, filtration, andreaction pads permit liquid to travel from the site of application tothe site of detection (i.e., the membrane), while simultaneouslyfiltering various solids and particles, colored materials, or othersubstances. The wash solution receiving pad can draw liquid from thereaction pad when wash solution is added to the membrane, thuspermitting the wash solution to carry away unwanted substances thatmight interfere with specific detection of the substance on themembrane.

The porous material serves various functions within each area or zone,and these functions significantly overlap with the functions that can beprovided in other areas of the device that comprise a porous material.For ease of description, many of the functions have been described withrespect to each particular area of the device; however, thecharacteristics should not be considered to be limited to only the areasfor which the characteristic has been described. The particular physicallocation of the porous material in differing embodiments will beapparent to those of skill in the art, and the particular functions ofthe porous material in each area will likewise be apparent. Thecharacteristics should not be considered limiting to that area. Forexample, in the receptacle region, the primary functions of the porousmaterial are to provide an area or volume for liquids containing orsuspected of containing a substance of interest to be retained, toprovide a reservoir for liquid to diffuse into and out of the porousmembrane comprising the specific binding pair member, and to draw liquidinto the reaction area. Thus, the porous material in this region issometimes referred to herein as the “reaction pad”. However, in certainembodiments, the reaction pad is also the sample receiving area, andthus also functions to receive, and typically filter, the sample aswell. Furthermore, due to placement of the membrane in contact with thereaction pad, the reaction pad serves as the initial acceptor for washsolution in embodiments where the membrane is washed. Accordingly, thereaction pad serves as an initial wash solution receiving pad as well.

Of course, other elements can be included in the device of the inventionto provide various advantages. All such additional elements are to beunderstood as encompassed by the present invention. Furthermore,additional method steps that provide additional advantages can beincluded, and are encompassed by the invention. Those of skill in theart are capable of including such elements and methods steps withoutundue experimentation and without departing from the full scope andspirit of the invention.

In embodiments, the device is provided in a kit. The kit can comprisethe device alone, in one or more copies of the same or multipledifferent various configurations. Alternatively, the kit may comprisesother materials, such as some or all of the materials, reagents, andequipment needed to practice at least one embodiment of the method ofthe invention.

The kits themselves can be fabricated from any suitable material, suchas cardboard, plastic, metal, or glass. Cardboard and plastic arepreferred materials for the kits. The kits are fabricated to suitablycontain all of the components provided by the kit. Thus, they aredesigned to be the appropriate size, shape, and strength for holding thevarious components selected to be provided by the kit.

The components provided by the kits can be in one or more containers.Containers of the device of the invention are described above, and canbe made of any suitable material, including any of the various plasticmaterials known to be useful in fabricating devices of this nature.Containers for other components can be made of any suitable material,including, but not limited to, plastic (e.g., a polymeric material),glass, metal, and rubber. The containers can be in any shape or form,and thus can be, for example, bottles, vials, cans, jars, or bags, suchas those made of metal, plastic, rubber, glass, or fabric. Thecontainers are preferably re-sealable or automatically sealing topreserve unused contents after initial opening.

Thus, in embodiments, the kits comprise one or more devices (e.g., 10,20, 25, 30, 50, 100) and at least one container containing a componentthat is useful for practicing at least one embodiment of the invention.For example, a kit can comprise 25 devices, sealed independently or twoor more together, in re-sealable pouches. Optionally, the pouches cancontain a dessicant to maintain low moisture content during storage. Thekit may also comprise any one or more of the following components, inany suitable container(s) and amounts/volumes: diluent (preferablyaqueous) for diluting an original sample; wash buffer (preferablyaqueous); specific binding pair member (e.g., a conjugate for binding tothe substance of interest in the sample, and which can be bound by anantibody spotted on the porous membrane of the device); substrate (e.g.,a substrate for an enzymatic reaction or otherwise for producing adetectable signal); positive control (e.g., an antigen of known identitythat binds with a known affinity to an antibody that is spotted on aporous membrane of the device); pipette (e.g., disposable pipettes foradding one or more reagent, etc. to the sample or device); test tube,gloves, applicator stick, pipette tips. Other optional components of thekits can be envisioned by those of skill in the art, and all such othercomponents are encompassed by the present invention.

Preferably, at least the devices are sterilized prior to, during, orafter insertion into the kits. Preferably, one, some, or all of theother components are sterilized prior to, during, or after insertioninto the kit. In highly preferred embodiments, each component in the kitis sterile or has been sterilized, either independently of one or moreof the other components, or together in the kit. Sterilization can beachieved by any known means, including, but not limited to, filtrationof liquids, irradiation by electromagnetic radiation (e.g., UV, gammairradiation), chemical sterilization (e.g., wiping with a disinfectantsuch as alcohol), and the like.

Instructions for using one or more components of the kit, or forpracticing the methods of the invention, may be included in the kit. Theinstructions may be provided as a separate component, such as printedmaterial on a paper, card, plastic sheet, or the like. Alternatively,the instructions may be provided on the kit itself, for example, on aside or the top or bottom of the kit. Alternatively, the instructionsmay be provided on a container for a component of the kit.

Turning now to the Figures, which depict various non-limiting specificembodiments of the invention, the elements described above are describedin various spatial relationships to each other, and use of variousconfigurations of the device of the invention to perform the method ofthe invention is described. It is to be understood that any dimensionsprovided in the Figures are provided as examples only, and that theactual sizes and shapes of the device are not limited to those providedin the Figures. For example, sizes can be on the order of a magnitude ormore larger or smaller than those exemplified in the Figures.Furthermore, it is to be understood that the Figures do not necessarilydepict all elements at proper scale with respect to each other, somebeing exaggerated for clarity purposes or for other reasons.

FIG. 1 depicts generally a basic configuration of the device 1 of thepresent invention. The device depicted in FIG. 1 comprises a receptacle100 that comprises a porous material in the form of a pad 110 (alsoreferred to herein as the “reaction chamber pad”). The device furthercomprises a porous membrane 120 that comprises a specific binding pairmember 130 for a substance of interest that is attached, by covalent,hydrophobic, or ionic bonding, along a single line in the center of themembrane. In practicing the invention with this configuration of thedevice, a liquid sample containing or suspected of containing asubstance of interest is applied to the receptacle 100 at any area. Theporous membrane 120 is then placed in direct contact with the porousmaterial 110 of the receptacle 100 such that the membrane 120 andmaterial 110 form a continuous contacting surface over at least the areaof the membrane 110 where the specific binding pair member 130 islocated. The liquid present in the porous material 110 is then permittedto diffuse into, out of, through, and around the membrane 120, enablingthe substance, if present, to contact the specific binding pair member130 and become specifically bound to it. In embodiments where thesubstance has already been labeled, detection of binding can beaccomplished at this point. In embodiments where a label has not yetbeen associated with the substance, the specific binding pairmember-substance complex can be labeled and then detected. Inembodiments, a wash step is included to reduce background. When a washis performed, the wash solution is applied to the membrane 120 andpermitted to soak into the porous material 110, thus removing materialsthat are not bound to membrane 120, and improving the signal-to-noiseratio. In embodiments, the substance in the sample is first bound by aspecific binding member that specifically binds to the one associatedwith the membrane (e.g., a conjugate), and the two specific binding pairmembers are allowed time to react.

The device 1 of FIG. 1 is also referred to at various points below as a“reaction chamber” when used in conjunction with other elements.

With respect to FIG. 2, a device 2 of the invention is depicted thatcomprises a receptacle 200 comprising a porous material 210. In thisembodiment, the porous membrane 220 is in contact with the porousmaterial 210 such that there is continuous contact between porousmembrane 220 and porous material 210 in the area of specific bindingpair member 230. In practice of the method of the invention with thisconfiguration of the device, a liquid sample containing or suspected ofcontaining a substance of interest is applied to the receptacle at anyarea other than the area in contact with porous membrane 220. The liquidpresent in the porous material is then permitted to diffuse into, outof, through, and around membrane 220, enabling the substance, ifpresent, to contact specific binding pair member 230 and becomespecifically bound to it. In embodiments where the substance has alreadybeen labeled, detection of binding can be accomplished at this point. Inembodiments where a label has not yet been associated with thesubstance, the specific binding pair member-substance complex can belabeled and then detected. In embodiments, a wash step is included toreduce background. When a wash is performed, the wash solution isapplied to membrane 220 and permitted to soak into porous material 210,thus removing materials that are not bound to membrane 220, andimproving the signal-to-noise ratio.

FIG. 3 depicts another embodiment of the device 3 of the invention, inwhich receptacle 300 comprising porous material 310 is contained incontainer 335, and wherein porous membrane 320 is held by holder 336. Inthis embodiment, holder 336 and container 335 are fabricated fromplastic. Holder 336 is integral with container 335, the two linked by arelatively flexible portion of the plastic at hinge 338. In otherembodiments, other materials and/or other structures are used,including, but not limited to use in providing the hinge function.

In practicing the method of the invention with this configuration of thedevice of the invention, a liquid sample containing or suspected ofcontaining a substance of interest is applied to the receptacle 300 atany area of the porous material 310, either directly or by way of thespace present between the receptacle 300 and the container 335, such asin area 331. Holder 336 comprising membrane 320 is then swung down byway of hinge 338 such that membrane 320 is in contact with porousmaterial 310, at least at a portion of membrane 320 where specificbinding pair member (not depicted) is present. Clamp or lip 337 engagesholder 336 to maintain membrane 320 in contact with material 310, asdescribed above. The liquid present in porous material 310 is thenpermitted to diffuse into and out of membrane 320, enabling thesubstance, if present, to contact specific binding pair member (notdepicted) present on membrane 320 and become specifically bound to it.In embodiments where the substance has already been labeled, detectionof binding can be accomplished at this point. In embodiments where alabel has not yet been associated with the substance, the specificbinding pair member-substance complex can be labeled and then detected.In embodiments, a wash step is included to reduce background. When awash is performed, the wash solution is applied to membrane 320 andpermitted to soak into porous material 310, thus removing materials thatare not bound to membrane 320, and improving the signal-to-noise ratio.

FIG. 4A depicts a device 4 of the invention in which the device 3 ofFIG. 3 is modified to include a wash solution receiving pad 440positioned in contact with porous material 410. Wash solution receivingpad 440 is fabricated from a porous material which can be the same ordifferent from reaction pad 410. In this embodiment, wash solutionreceiving pad 440 is a separate pad from reaction pad 410; however, inother embodiments, reaction pad 410 and wash receiving pad 440 are thesame element, the differentiation being based primarily on function.

More specifically, receptacle 400 comprising reaction pad 410 iscontained in container 435, which contains not only receptacle 400 butwash receiving pad 440 as well. Porous membrane 420 is held by holder436. In this embodiment, holder 436 and container 435 are fabricatedfrom plastic and holder 436 is integral with container 435, the twolinked by a relatively flexible portion of the plastic at hinge 438. Inother embodiments, other materials and/or structures are used, such asto provide the hinge function.

In practicing the method of the invention with this configuration of thedevice of the invention, a liquid sample containing or suspected ofcontaining a substance of interest is applied to the receptacle 400 atany area of reaction pad 410, either directly or by way of the spacepresent between the receptacle 400 and the container 435, such as inarea 431. Holder 436 comprising membrane 420 is then swung down by wayof hinge 438 such that membrane 420 is in contact with reaction pad 410,at least at a portion of membrane 420 where specific binding pair member(not depicted) is present. Clamp or lip 437 engages holder 436 tomaintain membrane 420 in contact with reaction pad 410, as describedabove. The liquid present in reaction pad 410 is then permitted todiffuse into and out of membrane 420, enabling the substance, ifpresent, to contact specific binding pair member (not depicted) presenton membrane 420 and become specifically bound to it. In embodimentswhere the substance has already been labeled, detection of binding canbe accomplished at this point. In embodiments where a label has not yetbeen associated with the substance, the specific binding pairmember-substance complex can be labeled and then detected.

In embodiments, a wash step is included to reduce background. When awash is performed, the wash solution is applied to membrane 420 andpermitted to soak into reaction pad 410, thus removing materials thatare not bound to membrane 420, and improving the signal-to-noise ratio.Typically, the amount of wash solution applied to membrane 420 exceedsthe holding capacity of pad 410, which has already been wetted (at leastpartially) with the liquid containing the substance of interest. In thissituation, excess wash solution (and some original liquid sample)travels through reaction pad 410 into wash solution receiving pad 440.

FIG. 4B depicts an alternative configuration of the device 4 of theinvention depicted in FIG. 4A, in which wash solution receiving pad 440is located beneath reaction pad 410. The principle of operation of thedevice is the same as described with respect to FIG. 4A. However, inthis configuration, it is not preferred that the liquid be applied inthe space between reaction pad 410 and container 435. In this Figure,all elements have the same identity as those in FIG. 4A.

FIG. 4C depicts an alternative configuration of the device depicted inFIG. 4A, in which wash solution receiving pad 440 and reaction pad 410are separated by removable, liquid-impermeable barrier 445. In thisconfiguration, impermeable barrier 445 is interposed between washsolution receiving pad 440 and reaction pad 410 and extends to theexterior of container 435 through slot 446 to expose tab 447. Practiceof the method of the invention with this configuration is similar tothat described above with respect to FIG. 4A. However, becauseimpermeable barrier 445 blocks migration of liquids into wash solutionreceiving pad 440, impermeable barrier 445 is typically removed aftercontacting of membrane 420 and reaction pad 410 and before applicationof wash solution to membrane 420. Use in this manner also restricts flowof the original sample from application/reaction pad 410 into washreceiving pad 440 until after diffusion of the sample in and out ofmembrane 420 has proceeding for a desired amount of time. All otherelements depicted in this panel are the same as those described withregard to FIG. 4A.

In another configuration of the device, depicted in FIG. 5, the basicdesign depicted in FIG. 1 is modified to produce a device 5 such thatthe porous material 510 of the receptacle 500 extends beyond thereaction chamber (i.e., is larger than the area of the porous membrane520) to provide a liquid sample application zone 550 comprising a sampleapplication pad 551. In the depicted embodiment, the sample applicationpad 551 and the reaction pad 510 are the same. However, in otherembodiments, these two elements are separate, and are arranged such thatthey are in physical contact with each other so that liquid may flowfrom one to the other. In practicing the invention with thisconfiguration of the device, a liquid sample containing or suspected ofcontaining a substance of interest is applied to the sample applicationpad 551 at any area within sample application zone 550. Due to itsporous nature, sample application pad 551 causes at least a portion ofthe applied sample to migrate to reaction pad 510, where it can thenmigrate, through passive diffusion, into and out of porous membrane 520,and allow the substance of interest, if present, to contact and bind tospecific binding pair member (not depicted) on porous membrane 520.Detection and washing, if desired, can be performed as described above.

FIG. 6 depicts device 6, which is a configuration of the device of theinvention as depicted in FIG. 5, but modified to include a container 635for the sample application pad 651 and reaction pad 610, and to includea holder 636 for porous membrane 620. In this particular embodiment,container 635 comprises an opening or port 655 that permits applicationof the liquid sample to application pad 651. Use of this embodiment ofthe device can proceed as discussed above.

FIG. 7 depicts device 7, which is a configuration of the device depictedin FIG. 6 in which a wash solution receiving pad 740 is providedadjacent to and in contact with reaction pad 710. In practice of theinvention with this configuration of the device, after sampleapplication to sample application pad 751, migration of the liquid toreaction pad 710 and diffusion of the liquid into and out of porousmembrane 720, a wash solution is added to membrane 720 and the excesswash solution (along with a portion of the original liquid sample) flowsthrough reaction pad 710 into wash solution receiving pad 740. Allelements depicted in this Figure other than those specificallyreferenced are the same as those in FIG. 6 and/or FIG. 5, where likeelements depicted are the same element from figure to figure.

FIG. 8 depicts device 8, which is a configuration of the device of theinvention in which reaction pad 810 is fabricated from the same materialas liquid sample application pad 851 and wash solution receiving pad840, but is compressed, as compared to sample application pad 851 andwash solution receiving pad 840, by pressure exerted by holder 836. Inthis preferred embodiment of the invention, pressure is exerted onmembrane 820, which causes compression of reaction pad 810. Thiscompression improves retention of sample in reaction pad 810, andpromotes diffusion of sample between reaction pad 810 and membrane 820.All elements depicted in this Figure other than those specificallyreferenced are the same as those in FIG. 7, FIG. 6, and/or FIG. 5, wherelike elements depicted are the same element from figure to figure.

FIG. 9 depicts device 9, which is a configuration of the device of theinvention in which reaction pad 910 is fabricated from the same materialas liquid sample application pad 951 and wash solution receiving pad940, but is compressed, as compared to sample application pad 951 andwash solution receiving pad 940, by pressure exerted from below (withrespect to porous membrane 920) by container 935. In the Figure,compression results from container 935, which is molded into a shapethat provides this effect. However, in other equivalent embodiments,container 935 comprises an additional element that provides the pressureon reaction pad 910. Exertion of pressure by container 935 provides thesame benefits described above with respect to FIG. 8 and as discussedother places in the description. All elements depicted in this Figureother than those specifically referenced are the same as those in FIG.8, FIG. 7, FIG. 6, and/or FIG. 5, where like elements are depictedand/or numbered the same from figure to figure.

FIG. 10 shows a configuration of device 10 of the invention, looking atthe outside of device 10 from the top. In this Figure, sampleapplication port 1055 is located at one end of device 10, and an openingin device 10 above porous membrane 1020 provides a detection window 1060through which detection of the presence of the substance of interest(and, optionally, one or more control reactions) can be viewed. Port1055 and window 1060 are located on container 1035. In practicing themethod using this embodiment of the device, sample is added to anapplication pad or reaction pad below or near port 1055, either directlyto the pad or to a space between the pad and container 1035. The sample,or a portion of it, travels along the pad at least until it comes intocontact with membrane 1020, preferably through a reaction pad (notdepicted) beneath membrane 1020. Diffusion of the sample in, out,across, and over membrane 1020 permits contact between the substance ofinterest in the sample and a specific binding pair member (not depicted)associated with membrane 1020. Detection of the substance bound to thespecific binding pair member on the membrane can occur by viewing themembrane through window 1060.

FIG. 11 shows an alternative configuration of the device 11 depicted inFIG. 10. In this configuration, sample application port 1155 is locatedat one corner of device 11, and detection window 1160 and membrane 1120are centrally located. In practice of the method of the invention withthis embodiment of the device, sample is added to an application pad orreaction pad (not depicted) beneath or near port 1155, either directlyto the pad or to a space between the pad and container 1135. The sample,or a portion of it, travels along the pad at least until it comes intocontact with membrane 1120. Diffusion of the sample in, out, across, andover membrane 1120 permits contact between the substance of interest inthe sample and a specific binding pair member associated with membrane1120. Detection of the substance bound to the specific binding pairmember on the membrane can occur by viewing the membrane through window1160.

FIG. 12 shows a top view of a configuration of device 12 of theinvention in which porous membrane 1220 comprises specific binding pairmember 1230, which is specific for the substance of interest, andpositive control 1270. In this configuration, both elements are locatedon membrane 1220 such that they are within the area defined by detectionwindow 1260. All of the elements depicted in FIG. 12 that are notspecifically discussed are the same as those depicted in FIG. 11, andall have the same function. Practice of the method of the inventionproceeds according to the disclosure above. Practice of the method usingthis embodiment of the device permits a single sample to be tested foran unknown substance while providing a positive control for performanceof the device and method.

FIG. 13 shows a configuration of device 13 of the invention in which asingle sample application pad 1351 within container 1335 bifurcates toconnect to two separate reaction pads 1310 a and 1310 b, each of whichis at least partially beneath and in contact with a different porousmembrane 1320 a and 1320 b and a different wash solution receiving pad1340 a and 1340 b. Porous membranes 1320 a and 1320 b comprise specificbinding pair members 1330 a and 1330 b, each of which are specific for adifferent substance. In this configuration of the device, the method ofthe invention can be used to detect two different substances in a singleliquid sample. In embodiments, one of the substances is known to bepresent in the sample (either naturally or as an added component), andthus one membrane (either 1320 a or 1320 b) acts as a positive controlfor the device and method.

FIG. 14A depicts a configuration of device 14 of the present inventionin which sample application pad 1451 extends beyond the interior area ofdevice 14 defined by container 1435. Sample application pad 1451 isintegral with filtration pad 1470, reaction pad 1410, and wash solutionreceiving pad 1440. Reaction pad 1410 is at least partially beneath andin direct contact with membrane 1420, which comprises specific bindingpair member 1430. In practice of the invention using this configurationof the device of the invention, application pad 1451 is contacted with aliquid sample by dipping into the liquid, inserting into a stream of theliquid (e.g., a stream of urine), application of the sample to the padby pipetting, or the like. The liquid sample passes through applicationpad 1451 into filtration pad 1470. In embodiments, a label (notdepicted) that binds to the substance of interest is present infiltration pad 1470 and is solubilized by the liquid. The label binds tosubstance that is present in the sample during passage throughfiltration pad 1470 (and/or at a later time during the assay). Theliquid sample then passes into reaction pad 1410 and diffuses into,through, out, and around membrane 1420. In embodiments, a label thatbinds to the substance of interest is present in and/or on the surfaceof reaction pad 1410 and is solubilized by the liquid. The label bindsto substance that is present in the sample during passage throughreaction pad 1410 (and/or at a later time during the assay). Diffusionfrom reaction pad 1410 into, through, out, and around membrane 1420permits contact of the substance of interest (if present) or thesubstrate-label complex with specific binding pair member 1430. Inembodiments where a direct label is used, it can either be bound to thesubstance at an earlier time (as described immediately above), or can bebound to the substance at the same time or after binding of thesubstance to the specific binding pair member. Upon formation of aspecific binding pair member-substance-label complex, the presence ofthe substance can be detected. In embodiments where an indirect label isused, the label can be added at any of the times described above. Uponformation of a specific binding pair member-substance-label complex, anyexcess label and other substances that might be present on the membranecan be washed away by applying a wash solution to membrane 1420. Thewash solution passes through membrane 1420 and into reaction pad 1410.Because there is excess wash solution beyond the carrying capacity ofreaction pad 1410, liquid is driven into wash solution receiving pad1440, filtration pad 1470, or both. Because wash solution receiving pad1440 is typically dry or substantially dry (due to selection of theappropriate amount of volume to be added at application zone 1451)whereas filtration pad 1470 is at least partially wet, wash solutionreceiving pad 1440 typically absorbs a majority of the wash solutionapplied to the membrane. The wash may be repeated as many times asnecessary to achieve a suitable signal-to-noise ratio. After washing,the substrate for the indirect label can be added, and another wash maybe performed, if desired, to reduce background signal. Detection of aspecific signal indicates the presence of the substance of interest inthe original sample.

FIG. 14B depicts another embodiment of device 14 depicted in FIG. 14A.In the device of FIG. 14B, applicator pad 1451 comprises an absorbentplastic material onto which a sample, such as urine, comprising anenzyme conjugate that binds to the substance of interest, is applieddirectly. The sample plus enzyme conjugate travels through filtrationpad 1470, which is unitary with application pad 1451, and enterscontainer 1435 through filtration pad 1470, which has been compressed bycontainer 1435. Sample plus enzyme conjugate travels through filtrationpad 1470 into reaction pad 1410, which is unitary with filtration pad1470 and is compressed by container 1435 in a similar fashion as withfiltration pad 1470. If a substance of interest is present, it willreact with the enzyme conjugate upon mixture before applying, duringpassage through sample application pad 1451, filtration pad 1470, orreaction pad 1410. The sample is permitted to contact membrane 1420 fora sufficient amount of time for the substance (or the substance-labelconjugate complex) to diffuse in, out, and through membrane 1420 and tocontact specific binding pair member 1430 (not shown) and form acomplex. Wash solution is then applied to membrane 1420 and excess washsolution travels through membrane 1420, and into at least wash solutionreceiving pad 1440. Detection of the presence or absence of thesubstance in the sample occurs by detecting a signal produced frommembrane 1420 at or near specific binding pair member 1430 (not shown).

FIG. 14C shows yet another configuration of device 14 depicted in FIGS.14A and 14B. In this configuration, sample application pad 1451,filtration pad 1470, reaction pad 1410, or a combination of two or allof these comprises a gold conjugate. The gold conjugate, whichspecifically binds to the substance of interest, is dissolved by theapplied liquid sample as it traverses the pads, and binds to thesubstance, if present. Upon contact of the liquid (now filtered) withmembrane 1420 via diffusion in, out, through, and about membrane 1420,the substance-gold conjugate binds to specific binding pair member 1430(not shown), resulting in generation of a detectable signal at or nearspecific binding pair member 1430 within about 30 seconds or more ofcontact of the liquid with membrane 1420. Presence of the substance inthe liquid is determined by detection of a signal (typically with theunaided eye) at detection window 1460 without the need for a washingstep.

FIG. 15 depicts a configuration of device 15 of the present invention.As shown in FIG. 15A, container 1535 is a “clam shell” type container inwhich a top half 1535 a and a bottom half 1535 b are attached to eachother along an edge by a flexible hinge 1580.

Although not depicted in the Figure, in embodiments, a holder for amembrane is integral with top half 1535 a and defines the edges of adetection window. A sample loading port is also integral to top half1535 a and is defined by an opening in top half 1535 a at a site abovean application pad. Top half 1535 a also comprises one or more pins orrecesses to accommodate pins, wherein engagement of the pins with therecesses causes a friction fit that holds top half 1535 a and bottomhalf 1535 b together.

In typical embodiments, bottom half 1535 b contains a sample applicationpad, a filtration pad, a reaction pad, and a wash receiving pad. It alsotypically comprises one or more recesses to accommodate pins or one ormore pins, wherein engagement of the recesses with the pins causes afriction fit that holds top half 1535 a and bottom half 1535 b together.

FIG. 15B depicts a top view of the bottom half of one embodiment ofdevice 15 depicted in FIG. 15A. In the Figure, sample application pad1551 is integral with filtration pad 1570, reaction pad 1510, and washsolution receiving pad 1540, all of which are 2.2 cm wide. Reaction pad1510 is 0.3 cm wider than sample application pad 1551, filtration pad1570, reaction pad 1510, and wash solution receiving pad, (0.15 cm oneach side) to completely support membrane 1520 (not depicted), whichmeasures 0.75 cm long by 2.5 cm wide). Friction fit alignment postrecesses 1581 are present as part of container 1535 b, as are clampposts 1582.

FIG. 15C shows a cross-section from the side of device 15 depicted inFIG. 15A and/or FIG. 15B, where top half 1535 a is positioned above, butnot in contact, with bottom half 1535 b, and where the hinge is removedto permit alignment of the top and bottom halves for descriptivepurposes. The Figure indicates the placement of sample application port1555 and viewing or detection window 1560.

FIG. 15D is a cross-section from the side of device 15 depicted in FIGS.15A, 15B, and/or 15C, in which the top half and bottom half are joinedby friction fit. As can be seen from the Figure, in this embodiment,connection of top half 1535 a with bottom half 1535 b results incompression of reaction pad 1510 at and near the area where membrane1520 is in contact with reaction pad 1510.

FIG. 16 depicts another embodiment of the device of the invention. InFIG. 16A, the device is depicted in its closed state. In FIG. 16B, thedevice is depicted in its open state. In this embodiment, device 16comprises a hinged container of plastic comprising top half 1635 a andbottom half 1635 b. Bottom half 1635 b comprises sample loading port1655, and contains a unitary combination of liquid sample applicationloading pad 1651, filtration pad 1670, and reaction pad 1610. Top half1635 a is attached to bottom half 1635 b by a flexible hinge 1680, whichis fabricated from the same plastic material as the rest of container1635. Top half 1635 a contains wash solution receiving pad 1640 andporous membrane 1620. Porous membrane 1620 comprises at least onespecific binding pair member (not depicted).

When in its closed position, membrane 1620 is sandwiched betweenreaction pad 1610 and wash receiving pad 1640, and is in contact withboth pads, contact being made between reaction pad 1610 and membrane1620 at least over a portion of membrane 1620 comprising at least onespecific binding pair member (not depicted). Sample is applied toapplication pad 1651 through application port 1655, and sample movesthrough application pad 1651 and filtration pad 1670 into reaction pad1610. The portion of the sample present in reaction pad 1610 diffusesinto and out of membrane 1620, and contacts the specific binding pairmember(s), where the substance of interest, if present, binds to thespecific binding pair member(s) and is retained on membrane 1620.

After a sufficient amount of time for reaction of the substance with thespecific binding pair member(s), top half 1635 a and bottom half 1635 bare separated by movement of the two about hinge 1680. If a direct labelis used, it may be added at this time, or it may have been present inreaction pad 1610, filtration pad 1670, or application pad 1651, andhave already bound to the substance of interest. When a direct label isused, detection of the presence of the substance may be made at thistime, or membrane 1620 may be washed to improve signal-to-noise ratio.If a wash is performed, the wash solution is added to membrane 1620, andthe wash solution is absorbed (after flowing through membrane 1620) bywash solution receiving pad 1640. If an indirect label is used, thelabel (or the substrate for the label, if the label has beenincorporated into application pad 1651, filtration pad 1670, or reactionpad 1610) is applied to membrane 1620 after separation of top half 1635a and bottom half 1635 b, and allowed to remain in contact with membrane1620 for a sufficient amount of time to react with the specific bindingpair member-bound substance. Wash solution is then applied to membrane1620 as described above. Where necessary, the substrate for the indirectlabel is then applied to membrane 1620 and allowed to remain in contactfor a sufficient amount of time for a complex between the label andlabel substrate to be formed, or for a detectable signal to begenerated.

FIG. 17 depicts a device 17, which shows an embodiment of the inventionin which a liquid impermeable plastic membrane 1745 is placed betweenreaction pad 1710 and wash solution receiving pad 1740 within container1735. In this embodiment, a liquid sample that has been clarified toremove large particulate material, and to which conjugate has beenadded, is applied to membrane 1720 and permitted to flow into reactionpad 1710, which is in contact with membrane 1720. Sufficient time isprovided for diffusion of the sample between reaction pad 1710 andmembrane 1720 such that the substance of interest, if present, binds toat least one specific binding pair member (not depicted). After asufficient amount of time, impermeable barrier 1745 is removed bypulling on tab 1747. Wash solution is applied to membrane 1720, andexcess wash solution and unbound substrate, conjugate, andconjugate-substrate complex is washed from membrane 1720 into reactionpad 1710 and then into wash solution receiving pad 1740. Detection ofthe presence of the substance is then accomplished by direct detectionof a signal from or near the specific binding pair member(s) (notdepicted), or by addition of substrate for the label. One or morefurther washings may be performed at this time.

FIG. 18 depicts another configuration of the device of the invention. Inthis configuration, device 18 comprises container 1835, which comprisessample port 1855 and sample application pad 1851, which are providedadjacent and connected to reaction pad 1810 and membrane 1820. Washreceiving pad 1840 is located below reaction pad 1810 and is separatedfrom reaction pad 1810 by impermeable membrane 1845. Removal ofimpermeable membrane 1845 by pulling on tab 1847 permits wash solutionreceiving pad 1840 and reaction pad 1810 to come in direct contact, andprovides a continuous flow from membrane 1820 to wash solution receivingpad 1840. In practice, this configuration of the device is used in asimilar manner as described with respect to FIG. 17, with the exceptionthat sample is added at sample application port 1855 rather than throughmembrane 1820.

FIG. 19 depicts an embodiment of the invention similar to that depictedin FIG. 16. FIG. 19A depicts the device in a closed position. FIG. 19Bdepicts the device in an open position. As can be seen in FIG. 19A,device 19 comprises a container comprising upper half 1935 a and lowerhalf 1935 b, connected via a hinge 1980. Upper half 1935 a comprisesreaction pad 1910, which is integral with sample application pad 1951,and which is held in place by holder 1936. Membrane 1920 is located inbetween reaction pad 1910 and wash receiving pad 1940, in directphysical contact with both through pressure exerted on membrane 1930 byholder 1936. Bottom half 1936 b comprises wash receiving pad 1940, indirect physical contact with membrane 1920. In practice of oneembodiment of the method of the invention with this configuration of thedevice, as depicted in the combination of FIG. 19A and FIG. 19B, sample(to which a label conjugate has been added) is applied to applicationpad 1951 (which is integral with reaction pad 1910 and filtration pad1970), and liquid of the sample is permitted to travel to reaction pad1910 and diffuse into and out of membrane 1920. Wash solution is addedto sample application pad 1951 and is drawn through reaction pad 1910and membrane 1920 by drawing of the liquid into wash solution receivingpad 1940 as a result of its being dry. Substrate for the label conjugateis then applied to membrane 1420 and the two halves 1935 a and 1935 bare separated by rotation about hinge 1980, thus revealing membrane1920. The presence of the substance is detected by visual or non-visualdetection methods by assaying for signal emitted from or near at leastone specific binding pair member (not depicted) spotted on membrane1920.

FIG. 20 is a configuration of device 20 of the invention comprisingcontainer 2035, and in which application pad 2051 is integral withreaction pad 2010, and in which application port 2055 is locatedabove/beneath and on the opposite side of membrane 2020 and detectionwindow 2060. Wash solution receiving pad 2040 is in direct contact withapplication pad 2051 and reaction pad 2010 and is located to the side ofthese pads, with respect to application port 2055 and detection window2060. In practice of an embodiment of the method of the invention withthis configuration of the device, sample (to which conjugate has beenadded) is applied to application pad 2051 through application port 2055,and sample is allowed to traverse into reaction pad 2010, and diffusebetween reaction pad 2010 and membrane 2020, which comprises at leastone specific binding pair member (not depicted). Device 20 is invertedafter the liquid sample has been fully or substantially fully drawn intoapplication pad 2051 and reaction pad 2010, and a desired amount ofdiffusion between pad 2010 and membrane 2020 has occurred. Wash solutionis applied to membrane 2020, and drawn through reaction pad 2010 andapplication pad 2055 into wash receiving pad 2040. Substrate for theconjugate is added and detection of the presence of the substance ofinterest in the liquid is performed, in accordance with known proceduresand the disclosure above. As with all other embodiments where anindirect label is used, incubation of the substrate and the label ispreferred to obtain optimal signal intensity.

FIG. 21 depicts one embodiment of the device of the invention, which isalso referenced in an Example below. In general, the embodimentcomprises the elements discussed above with regard to other embodiments.In one particular embodiment of the device depicted in FIG. 21, thedevice comprises a reaction window opening onto a porous membranecomprising two lines of immobilized antibodies spotted thereon. Forexample, it can contain a test line (or “T” line), having antibodiesagainst C. difficile toxin A, toxin B, or both. It can also contain asecond line (or “C” line) to act as an internal control, for examplehaving anti-IgG antibodies or other antibodies specific for otherantigens. In use, the device can detect the presence of a substance ofinterest, such as toxin A and/or B, in a sample. For example, a samplecan be added to a tube containing a mixture of a diluent (e.g., abuffered protein solution containing 0.02% thimerosal) and conjugate(e.g., a mouse monoclonal antibody specific for toxin A coupled tohorseradish peroxidase and a goat polyclonal antibody specific for toxinB coupled to horseradish peroxidase in a buffered protein solutioncontaining 0.02% thimerosal). The diluted sample-conjugate mixture canthen be added to a sample well, which is an opening in the shell of thedevice, and which opens onto a porous material (filter pad) in an areafor receiving the sample, which is distinct from the reaction window.After adding the diluted sample to the porous material through thesample well, the device can be incubated at a suitable temperature, forexample at room temperature, for a sufficient amount of time, forexample 15 minutes. During this incubation period, the substance ofinterest (e.g., toxin A and/or B), if present in the sample, binds tothe conjugate (e.g., anti-toxin antibody-peroxidase conjugate). Afterapplication of the sample to the device, the substance-conjugate (e.g.,toxin-antibody) complexes, if present, migrate through at least oneporous material to the porous membrane containing the immobilizedantibodies. A sufficient amount of time (e.g., one minute) is providedfor diffusion of the complexes in and out of the porous membrane.Complexes, if present, are captured by the immobilized antibodies on theline(s). The porous membrane, at least at the area comprising a portionof the lines, can then be optionally washed with a wash buffer (e.g., abuffered solution containing 0.02% thimerosal). The device can then bedeveloped with the addition of a substrate (e.g., a solution comprisingtetramethylbenzidine). After an incubation period (for example, 10minutes), the presence of a complex at the test line can be determinedby, for example, visually examining for the appearance of a line (e.g.,a blue line) at the area where the “T” line is present on the porousmembrane below the reaction window. A line at this area indicates apositive test. Where a line (e.g., blue line) exists at the “C” area ofthe porous membrane below the reaction window, a positive controlreaction has occurred, indicating that the device and method are workingproperly, and that the results (presence or absence of substance ofinterest) are valid. Where a control is run, the control can include anappropriate antigen for an antibody spotted onto the membrane at the “C”line, such as an antigen in a buffered aqueous solution. FIG. 21A-Ddepict the lines described herein, and note various possible results.

Thus, the invention provides a method of detecting at least onesubstance of interest in a liquid sample, where the method comprises:providing a liquid sample comprising or suspected of comprising thesubstance(s) of interest; applying the liquid sample to a porousmaterial in a sufficient amount to at least partially wet the porousmaterial; contacting the porous material with a porous membranecomprising at least one specific binding pair member that is capable ofbinding, either directly or indirectly, the substance(s) of interest;maintaining the porous material and porous membrane in contact for asufficient amount of time for liquid present in the porous material todiffuse in, out, through, and/or about the porous membrane, whereindiffusion of the liquid in, out, through, and/or about the porousmembrane results in contact of the substance(s) of interest, if present,to be bound, either directly or indirectly, to the specific binding pairmember(s); and detecting the presence or absence of a complex comprisingthe specific binding pair member(s) and the substance(s) of interest,wherein the presence of at least one complex indicates the presence ofat least one of the substances of interest in the liquid sample. Inembodiments, the method can further comprise providing a devicecomprising the porous material and porous membrane. In embodiments, theporous material filters the liquid sample to remove substances having asize greater than a pre-determined value. In embodiments, the filteringis by way of discontinuous wicking of liquid from the liquid samplethrough the porous material. In embodiments, the method can be used inconjunction with a liquid that comprises two or more substances ofinterest, and one, two, or more of these substances can be detectedusing a single device and/or a single practice of the method of theinvention. Thus, in certain embodiments of the method, each substance ofinterest is different than each other substance of interest, and themethod detects one, two, or more of them. The method can be practiced onliquid samples containing feces, blood, food, or an environmental sample(e.g., a toxic substance in ground water). In exemplary embodiments, themethod detects one or both of Clostridium difficile toxin A andClostridium difficile toxin B. In embodiments, the substance of interestis one or more toxin, bacterium, virus, bacterial product, enzyme (e.g.,prokaryotic, eukaryotic), or parasite. In some embodiments, thesubstance of interest is glutamate dehydrogenase. It can also be ananimal or human product, an antibody, or lactoferrin.

The method can be practiced using one or more specific binding pairmembers. In embodiments, one or more of the specific binding pairmember(s) is an antibody, wherein each of the antibodies is eitherdifferent or the same as one or more others.

The general method can further comprise washing the membrane prior todetecting the presence of a complex.

In embodiments, applying the liquid sample to the porous materialcomprises applying the liquid sample at a location on the porousmaterial that is spatially separated from the porous membrane, wherebyat least the liquid of the liquid sample travels into the porousmaterial and then the porous membrane. In certain embodiments, liquidsample that is applied at a region of a sample loading zone that isdistant from a detection zone travels through the porous material to theporous membrane through a wicking process. In embodiments, a physicalforce is applied to the membrane, the porous material, or both, and sucha force improves sensitivity of the device and method of the invention.

The method of the invention comprises detecting a signal to determinethe presence of a substance of interest. In embodiments, detectingcomprises observing a signal emitted from a label bound to the substanceof interest. In particular embodiments, the signal is produced by acolored precipitating product that forms in or around the specificbinding pair member. Detecting thus can be through detection of acomplex. Therefore, the method can comprise combining a labeledconjugate with the liquid sample prior to applying the liquid sample tothe porous material. The labeled conjugate can comprise a latex bead orother colored particle, a colloidal gold particle, or a reactivesubstance that binds to a substrate to create a detectable signal. Insome embodiments, the signal is a non-visual signal.

The invention includes a device for detecting at least one substance ofinterest in a liquid sample. In embodiments, the device comprises: (a) areceptacle comprising a porous material for receiving the liquid sample,wherein the porous material is capable of absorbing and transmitting atleast a portion of the liquid sample, and (b) a porous membrane thatcomprises a specific binding pair member that is specific for thesubstance of interest or a substance bound to the substance of interest,wherein the receptacle and porous membrane are each shaped to permit theporous membrane to be in direct contact with the porous material over atleast a portion of the porous membrane that comprises the specificbinding pair member. The device can comprise a container containing thereceptacle, a holder for the porous membrane, a wash solution receivingpad, a liquid sample application pad, a filtration pad, or two or moreof these elements. Each element may be subdivided into two or morefunctional zones, which, while optionally being fabricated of the samematerial, can be fabricated from different materials than one or more ofthe other zones.

In embodiments, the device comprises a container containing a reactionpad comprising the porous material and the porous membrane, thecontainer comprising a holder for the porous membrane, wherein thecontainer causes pressure to be exerted on the porous membrane and/orthe porous material such that at least a portion of the porous materialis compressed. Of course, the container can contain other elements, asdiscussed above. Compression of the porous material can cause the porousmembrane and the porous material to be in direct contact over at least aportion of the porous membrane, and can improve the function of thedevice and method of the invention. For example, the pressure can permitone or more liquids to pass between the porous membrane and the porousmaterial by passive diffusion.

The invention thus provides a device for detecting at least onesubstance of interest in a liquid sample, where the device comprises:(a) a receptacle comprising a porous material for receiving the liquidsample, wherein the porous material is capable of absorbing andtransmitting at least a portion of the liquid sample, and (b) a porousmembrane that comprises a specific binding pair member that is specificfor the substance of interest or a substance bound to the substance ofinterest, wherein the receptacle and porous membrane are each shaped topermit the porous membrane to be in direct contact with the porousmaterial over at least a portion of the porous membrane that comprisesthe specific binding pair member, wherein the porous membrane and porousmaterial are different elements having a different chemicalconstitution. In embodiments, the porous membrane and porous materialare in physical contact such that liquid sample applied to the porousmaterial diffuses in, out, through, and about the porous membrane. Inaddition, the device may be constructed such that physical contactbetween the porous membrane and porous material is created such that thesensitivity of the device is improved. The device can be configured suchthat the porous material and porous membrane are in contact with eachother such that the substance of interest does not need to traverse theporous membrane in a unidirectional manner for the device to detect thesubstance of interest. In exemplary embodiments, the porous material andporous membrane are in contact with each other in such a way to permitsimple, non-directional diffusion of a liquid between the two to occur.

The invention thus provides a device for detecting the presence oramount of a substance of interest in a liquid sample, where the devicecomprises: a sample receiving zone for receiving the liquid sample,wherein the sample receiving zone is present on a porous material; asample filter zone for filtering the liquid sample received at thesample receiving zone, wherein the sample filtering zone is present on aporous material; a porous membrane comprising a specific binding memberat a detection zone that specifically binds to the substance of interestor a substance bound to the substance of interest, wherein the porousmembrane is not the same element as any of the porous materials, andwherein the porous material and porous membrane are in physical contactover at least an area comprising a portion of the detection zone, andwherein the porous material and porous membrane are in physical contactin a configuration that permits liquid present in the liquid sample todiffuse in, out, through, and about the porous membrane in asubstantially random, non-directional manner in an area comprising atleast a portion of the detection zone. Of course, one or more zones canbe present on a single porous material or on two or more differentmaterials. Likewise, they can be present on two or more differentmaterials, each independently selected to have the same or differentcomposition as one or more other. As mentioned above, the device maycomprise a container that contains at least a portion of the porousmaterial(s) and porous membrane. In embodiments, the device comprises aporous material comprising a wash solution receiving zone.

Other exemplary embodiments of the device of the invention, and use ofthe device in practice of the method of the invention, are provided inthe Examples that follow and others will be apparent from thedescription and drawings.

EXAMPLES

The invention will be further explained by the following Examples, whichare intended to be purely exemplary of the invention, and should not beconsidered as limiting the invention in any way.

Example 1 Use of an Embodiment of the Device and Method of the Invention

This Example details a typical in vitro use and guidelines for in vitrouse of an embodiment of the method of the invention in an embodiment ofthe device of the invention, as depicted in FIGS. 21A-D, in whichClostridium difficile Toxins A and B are detected. The protocolgenerally follows the protocol provided in the TOX A/B QUIK CHEK™ kit(TechLab, Blacksburg, Va.; cat. no. T5033), which is incorporated hereinin its entirety by reference. Unless otherwise noted, the protocolprovided in the TOXA/B QUIK CHEK™ kit was use in the Examples. Generalguidelines are provided in this Example and in the TechLab kit, but arenot necessarily applicable to other embodiments of the method of theinvention.

Collection and Handling of Fecal Specimens

Standard collection and handling procedures used for fecal specimens areappropriate. Specimens should be stored between 2° C. and 8° C. It ispreferred to test specimens that are less than 24 hours old. It ispreferred to store specimens frozen (less than or equal to −10° C.) ifthe test cannot be performed within 72 hours of collection. While datashows that one freeze-thaw cycle does not harm the sample for use withC. difficile toxins A and B, it is noted that freezing and thawing of aspecimen, especially multiple times, might result in loss of activitydue to degradation of the toxins. Fecal specimens that have beenpreserved in 10% Formalin, MF, SAF, or PVA, or specimens that are intransport media such as Cary Blair or C&S typically do not give asoptimal results as fresh samples or those preserved in othercompositions.

Specimens should be thoroughly mixed (e.g., vortexed) prior toperforming the assay. Storing of fecal specimens in the diluent is notrecommended. It is preferred that one immediately test a sample once thefecal specimen is diluted in diluent. Disposable pipettes graduated at50, 100, 200, and 300 ul may be used.

Sample Preparation

optionally bring all reagents and devices to room temperature beforeuse.

Set up one cassette (device) for each specimen to be tested.

Add 0.4-0.6 ml (e.g., 0.425 ml or 0.5 ml) diluent to each dilution tubeusing a plastic dropper.

Evenly suspend (e.g., vortex) the specimens before transferring. ForLiquid/Semi-solid specimens, draw the specimen halfway to the first markfrom the end (25 ul). Dispense the specimen into the diluent. Use thesame pipette to mix the diluted specimen by gently aspirating, thendispensing the mixture several times. For Formed/Solid specimens, mixthe specimen thoroughly. Using a wooden applicator stick, transfer asmall portion (approximately 2 mm diameter) of the specimen into thediluent. Emulsify the specimen using the applicator stick. As anoptional control, add 1 drop of positive control or negative control(specimen diluent) to tubes containing 0.4 ml diluent.

Add 1 drop of conjugate to the diluted specimen and mix the tubecontents by vortexing.

Test Procedure

Obtain the required number of cassettes, one per specimen, and one perpositive or negative control. Label the membrane cassettesappropriately.

Obtain the prepared samples. Using a disposable transfer pipette,transfer 300-400 ul of the diluted sample-conjugate mixture into thesample port of the cassette and incubate the cassette at roomtemperature for 15 minutes. An increasing wet area will be visible inthe results window. If no wet area appears in the results window, add100 ul of diluent to the sample port and wait an additional 5 minutes.

After 15 minutes, add 300 ul of wash buffer to the reaction port. Allowthe wash buffer to enter the reaction port completely.

Add 2 drops of substrate to the reaction port and allow the cassette toincubate at room temperature for 10 minutes. At the end of 10 minutes,read the results from the detection window. Observe for the appearanceof a colored (e.g., blue) line representing the control line (see FIG.21A). The lines may appear faint to dark in color.

Interpretation of Results

Positive Result (FIG. 21A): Two lines are visible, one on the bottom ofthe reaction port (control line) and one on the top of the reaction port(test line). A positive result indicates the presence of C. difficiletoxin and a properly reactive control.

Negative Result (FIG. 21B): A single control line is visible only on thebottom of the reaction port. No test line is visible on the top of thereaction port. A negative result indicates the absence of C. difficiletoxin but a properly reactive control.

Invalid Result (FIGS. 21C and 21D): All completed reactions should havea visible control line on the bottom of the reaction port. The test isinvalid if a control line is not present on the completed cassette.

Example 2 Comparison of Detection of Clostridium difficile Toxins A andB Using the Device of the Invention and Tissue Culture

An embodiment of the device of the invention was used to detect acombination of C. difficile toxin A and toxin B in 50 fecal samples, andthe results compared to results obtained for the same samples usingtissue culture methods. Tissue culture detection of C. difficile toxinin fecal samples is the art-recognized assay of choice because it isconsidered to be the most sensitive method for detecting the toxins. Themethod described in Example 1 was used to detect the toxins.

The tissue culture test was the C. difficile Tox-B Test kit manufacturedby Tech Lab, Inc. (cat. no. T5003), and the procedure was as describedin the product insert. In brief, fecal samples were diluted 1:10 indiluent and filtered through a 0.45 micron sterile filter. Each fecalsample was added (50 microliters) to each of two tissue culture wells.One well received 50 microliters of antitoxin to neutralize C. difficiletoxins A and B and the other well received 50 microliters of onlyphosphate buffered saline. The human foreskin tissue cultured cells wereincubated at 37° C. for 24 hours then examined for rounding of thecells, and examined again at 48 hrs. Wells in which greater than 50% ofthe cells were rounded were considered positive. For a positive overallreaction, the well containing antitoxin had to be normal while the wellwithout antitoxin showed rounding of the cells.

Table 1 shows the results of the assays, and compares the results of themethod and device of the present invention with the tissue cultureassay.

TABLE 1 N = 50 A/B Invention pos A/B Invention neg Tiss cult pos 8  0Tiss cult neg 1 41 Sensitivity 88.9 Specificity 100.0 Pred Pos Val 100.0Pred Neg Val 97.6 Correlation 98.0

The results indicate that a device and method of the present inventionperform almost identically to tissue culture.

Example 3 Comparison of Detection of Clostridium difficile Toxins A andB Using the Device of the Invention and ELISA

A device of the invention was used to detect a combination of C.difficile toxin A and toxin B in 50 fecal samples, and the resultscompared to results obtained for the same samples using ELISA. Themethod described in Example 1 was used to detect the toxins.

The TechLab Inc. Tox A/B Test kit was used in this experiment accordingthe directions in the product insert. Briefly, feces were diluted 1:5 insample diluent and 100 microliters were added to wells in an ELISA 96well plate. Then 50 microliters of conjugate solution (containingantibodies to C. difficile toxins A and B that had been conjugated tohorseradish peroxidase) was added to each well. Wells were incubated 50min at 37° C. and then the wells were washed to remove horse radishperoxidase conjugate that had not bound to toxins (that had bound toantibodies coating the wells). This sandwich of antibodies and enzymewas then detected by addition of 100 microliters of substrate solutionwith incubation for 10 minutes followed by addition of 50 microliters ofdilute acid to stop the reaction. Positive reactions were those wellswith an Optical Density at 450 nm of greater than 0.12.

Table 2 shows the results of the assays, and compares the results of themethod and device of the present invention with the ELISA assay.

TABLE 2 N = 50 A/B Invention pos A/B Invention neg Tiss cult pos 8  1Tiss cult neg 1 41 Sensitivity 88.9 Specificity 97.6 Pred Pos Val 88.9Pred Neg Val 97.6 Correlation 96.0

The results indicate that the device and method of the present inventionproduce results that are comparable to the sensitive ELISA method used.

Example 4 Investigation of Relative Sensitivity of a Device and Methodof the Invention

The sensitivity of a device and method of the invention was determined.The device and method used were that of the TOX A/B QUIK CHEK™ test fortoxins A and B (TechLab, Inc.). Briefly, the sensitivity of the deviceand method was determined using serial two-fold dilutions of highlypurified toxins A and B.

The test was consistently positive at a concentration of 0.63 ng/mL fortoxin A and 1.25 ng/mL for toxin B. The results of six separate tests(Tests 1 through 6) with serially diluted toxin A or toxin B for thetest are shown in the tables below.

TABLE 3 Reaction of highly purified toxin A in the TOX A/B QUIK CHEK ™test Conc. (ng/ml) Test 1 Test 2 Test 3 Test 4 Test 5 Test 61.25 + + + + + + 0.63 + + + + + + 0.32 + + − + +/− +/− 0.16 − − +/− − −− 0.08 − − − − − −

TABLE 4 Reaction of highly purified toxin B in the TOX A/B QUIK CHEK ™test Conc. (ng/ml) Test 1 Test 2 Test 3 Test 4 Test 5 Test 61.25 + + + + + + 0.63 − − − − + + 0.32 − − − − − − 0.16 − − − − − − 0.08− − − − − −The data presented in Tables 3 and 4 are representative of theparticular results obtained in the particular testing runs performed. Inother tests, sensitivity of 0.16 for Toxin A and 0.32 to 0.63 for ToxinB has been often seen.

Example 5 Reproducibility and Precision of an Apparatus and Method ofthe Invention

To determine the reproducibility and precision of devices and methods ofthe invention, an embodiment of the device of the invention, as depictedin FIG. 21, was testing using an embodiment of the method of theinvention, according to a protocol supplied with the TechLab TOX A/BQUIK CHEK™ test. More specifically, a total of 8 fecal specimens, 6positive and 2 negative, were tested in three different laboratoriesusing the TOX A/B QUIK CHEK™ test (TechLab, cat. no. T5033) according tothe manufacturer's instructions. To challenge the cutoff, 2 weaklypositive specimens that gave faint lines when analyzed by the inventorswere included in the 6 positive specimens. All of the specimens wereclassified by a predicate device, the C. DIFFICILE TOX A/B II™ test(TechLab; cat. no. T5003), which is widely accepted as a highlysensitive and accurate test for the presence of C. difficile toxins Aand B. All specimens were kept frozen at ≦−10° C. until the assay wasperformed. Each of the laboratories tested the specimens on 3 differentdays. The results from each laboratory were subsequently submitted tothe inventors and compared with the inventors' own results. The results,shown below, were consistent among the different locations, andexhibited a correlation of 100%. The positive specimens were confirmedto be positive and the negative specimens were confirmed to be negativeat all sites using the TOX A/B QUIK CHEK™ test.

TABLE 5 Reproducibility/Precision Testing By Inventors Of Fecal SamplesUsing A Device And Method According To The Invention Specimen Code TOXA/B II ™ (n = 8) ELISA Day 1 Day 2 Day 3 TL001 + + + + TL002 + + + +TL003 + + + + TL004 + + + + TL005 + + + + TL006 + + + + TL007 − − − −TL008 − − − − Percent N/A 100 100 100 Correlation

TABLE 6 External Reproducibility/Precision Testing Of Fecal SamplesUsing A Device And Method According To The Invention Specimen Code TOXA/B II ™ (n = 8) ELISA Day 1 Day 2 Day 3 TL001 + + + + TL002 + + + +TL003 + + + + TL004 + + + + TL005 + + + + TL006 + + + + TL007 − − − −TL008 − − − − Percent N/A 100 100 100 Correlation

TABLE 7 External Reproducibility/Precision Testing Of Fecal SamplesUsing A Device And Method According To The Invention Specimen Code TOXA/B II ™ (n = 8) ELISA Day 1 Day 2 Day 3 TL001 + + + + TL002 + + + +TL003 + + + + TL004 + + + + TL005 + + + + TL006 + + + + TL007 − − − −TL008 − − − − Percent N/A 100 100 100 Correlation

TABLE 8 External Reproducibility/Precision Testing Of Fecal SamplesUsing A Device And Method According To The Invention Specimen Code TOXA/B II ™ (n = 8) ELISA Day 1 Day 2 Day 3 TL001 + + + + TL002 + + + +TL003 + + + + TL004 + + + + TL005 + + + + TL006 + + + + TL007 − − − −TL008 − − − − Percent N/A 100 100 100 CorrelationAs can be seen, the device and method performed well in the hands offour different practitioners.

Example 6 Effect of Freeze-Thaw on Specimens

To further characterize devices and methods according to the invention,an embodiment of the device was used in conjunction with a methodaccording to the invention to determine the suitability of each withspecimens that had been subjected to at least one freeze-thaw cycle.

A total of eight fecal specimens, consisting of 6 positive and 2negative specimens, were tested using an embodiment of the device of theinvention, as depicted in FIG. 21, and an embodiment of the method ofthe invention, both of which are available in the TOX A/B QUIK CHEK™test from TechLab, Inc. (cat. no. T5033) before and after a singlefreeze-thaw cycle. The specimens had been tested previously in the C.DIFFICILE TOX A/B II™ (TechLab, Inc.; cat. no. T5003) test for thepresence or absence of toxins A and B. The results are shown in thetable below. Included is the residual reactivity in the C. DIFFICILE TOXA/B II™ test after the freeze-thaw cycle. The results showed that thepositive specimens remained positive after the freeze-thaw cycle and thenegative specimens remained negative. No conversion ofpositive-to-negative or negative-to-positive was observed in any of thespecimens.

TABLE 9 Effect of Freeze-Thaw Cycle on Device and Method of theInvention Specimen TOX A/B II ™ INVENTION TOX A/B II ™ INVENTION CodeELISA (before ELISA (after (n = 8) (before freezing freezing) (afterfreezing) freezing) TL001 + + + + TL002 + + + + TL003 + + + +TL004 + + + + TL005 + + + + TL006 + + + + TL007 − − − − TL008 − − − −

Example 7 Effect of Specimen Storage Between 2° and 8° C. For 72 Hours

To further investigate the use of the device and method of the inventionfor detection of substances of interest in samples, six positive and twonegative fecal specimens (with respect to C. difficile toxins A and B)were tested at times 24, 48, and 72 hours using a device and methodaccording to the present invention, specifically in the TOX A/B QUIKCHEK™ test (TechLab, Inc.; cat. no. T5033) according to themanufacturer's instructions, to evaluate the stability of the toxins infecal samples. The results, shown below, demonstrate that the device andmethod performed consistently at each time interval. In addition, itshows that the C. difficile toxins are stable for at least 72 hoursunder these test conditions. All of the positive specimens remainedpositive and the negative specimens remained negative at each timeperiod.

TABLE 10 Effect of Specimen Storage Between 2° and 8° C. for 72 HoursDay 1 Day 3 C. C. difficile difficile TOX Day 2 TOX A/B Day 1 C. Day 2A/B Day 3 Speci- II ™ TOX A/B difficile TOX A/B II ™ TOX A/B mens 20 minQUIK TOX QUIK 20 min QUIK (n = 8) assay CHEK ™ A/B II ™ CHEK ™ assayCHEK ™ TL001 + + + + + + TL002 + + + + + + TL003 + + + + + +TL004 + + + + + + TL005 + + + + + + TL006 + + + + + + TL007 − − − − − −TL008 − − − − − −

Example 8 Use of a Method and Apparatus to Test Clinical Samples in aClinical Lab Setting

The method and apparatus used in Examples 4-7, above, were used toanalyze clinical samples suspected of containing C. difficile toxins Aand/or B. More specifically, an embodiment of the device of theinvention, coupled with an embodiment of the method of the invention,sold together by TechLab, Inc. under the tradename TOX A/B QUIK CHEK™(TechLab cat. no. T5033) was compared with tissue culture assays at 3commercial clinical laboratories and by the inventors. The study sitesand investigators, along with the number and source of specimens arepresented in the following table. The device and method of the inventionwere compared to tissue culture assay because tissue culture assay isconsidered the “Gold Standard” for detecting C. difficile toxin in fecalsamples. Discrepant results were analyzed using either the C. difficileTOX A/B II™ test or the Meridian Premier™ Toxins A&B test, both of whichare microtiter ELISAs for detecting toxins A and B in fecal specimens.For studies performed by the inventors in this Example, tissue cultureassay was performed using the C. DIFFICILE TOX-B TEST assay of TechLab,Inc.

When comparing the device and method of the invention to the tissueculture assay the sensitivity, specificity, positive and negativepredictive values, and percent correlation were determined. The 95%Confidence Intervals were also determined for the analysis versus tissueculture assay.

The gender identification was available for 294 patients. There were 177females (60.2%) and 117 males (39.8%). Age information was available for613 patients. The age ranged from approximately 1 year to 95 years, withthe distribution shown in the table illustrated in FIG. 22. The numberabove each bar in the table represents the number of patients who werein the specified age group.

The tables below show a summary of the clinical performance of thedevice and method of the invention. Results from all 5 clinical studiesperformed are included in the summary. Results from the device andmethod of the invention were compared to tissue culture assay anddiscrepant results were analyzed by either the C. DIFFICILE TOX A/B II™test (the presently discussed device and method of the invention) or theMeridian Premier™ Toxins A&B test. The results show that the TOX A/BQUIK CHEK™ test exhibited a sensitivity and specificity of 90.2% and99.7%, respectively, compared to the tissue culture assay. Thepredictive positive and negative values were 98.6% and 97.9%,respectively, and the correlation was 98.0%.

TABLE 11 Summary of Clinical Performance of a Device and Method of theInvention Tissue Culture Tissue Culture n = 842 Positive SamplesNegative Samples TOX A/B QUIK CHEK ™ 138  2 positive TOX A/B QUIK CHEK ™15 687 negative 95% Confidence Interval Sensitivity 90.2 84.1-94.2Specificity 99.7 98.8-99.9 Predictive Positive Value 97.9 96.4-98.7Correlation 98.0 97.8-98.2

Of the 2 tissue culture-negative/TOX A/B QUIK CHEK™-positive samples, 1was negative in the TOX A/B II™ test. Of the 15 specimens that weretissue culture-positive/TOXA/B QUIK CHEK™-negative, 12 were negative inthe C. DIFFICILE TOX A/B II™ test or the Meridian Premier™ Toxins A&Btest.

Example 9 Effect of Fecal Specimen Consistency

To further characterize the method and device of the invention, anembodiment of the device was tested with an embodiment of the method todetermine the effect of fecal specimen consistency on the performance ofthe device and method.

The reaction of fecal specimens of varying consistencies in the TOX A/BQUIK CHEK™ test is shown in the table below. A total of 805 fecalsamples of known consistency were included in the analysis. Thepercentages of positive reactions using either tissue culture assay orthe TOX A/B QUIK CHEK™ test were similar in all three types of fecalspecimens (liquid, semi-solid, and solid). All of the specimens weresubmitted for C. difficile testing. The basis of the submission was theclinical history of the patient and not the consistency of the specimen.The results show the TOX A/B QUIK CHEK™ test performed similarly to thetissue culture assay when testing samples of different consistencies.

TABLE 12 Reaction of fecal specimens of varying consistencies in the TOXA/B QUIK CHEK ™ test Semi-solid # of Specimens Liquid SpecimensSpecimens Solid Specimens (n = 805) (n = 487) (n = 294) (n = 24)Positive by tissue 87 (17.9%) 56 (19.0%) 3 (12.5%) culture assayPositive by TOX A/B 76 (15.6%) 50 (17%)   3 (12.5)   QUIK CHEK ™

Example 10 Comparison of Detection of Clostridium difficile GlutamateDehydrogenase Using the Device of the Invention and ELISA

The device of the invention was used to detect the glutamatedehydrogenase antigen of C. difficile in 49 fecal samples, and theresults compared to results obtained with an ELISA method. The methoddescribed in Example 1 was used to detect the toxins, with the followingmodifications.

Fecal samples for use in the device were diluted as specified in Example1 with sample diluent containing antibodies (in this case specific forthe glutamate dehydrogenase enzyme of C. difficile) that had beenchemically conjugated to horseradish peroxidase detecting enzyme(conjugate). The mixed sample (300 microliters) was then applied to theapplication pad (wicking pad) through the hole in the device(application port) and after 15 minutes at room temperature, washingsolution (saline/detergent mix) was added to the top of the membranefollowed by the chemical substrate solution. The results were readvisually as specified in Example 1.

The TechLab Inc. Tox A/B ELISA was used as specified in themanufacturer's product insert. Briefly, the fecal sample was diluted 1:5in sample diluent and mixed by vortexing. Each well of the ELISA platereceived 50 microliters of conjugate solution containing antibodiesspecific for glutamate dehydrogenase coupled to horseradish peroxidaseand then 100 microliters of a mixed sample was added to each well. Theplate was then incubated at 37° C. for 50 minutes to allow theantibodies attached to the microwells and antibody in the conjugatesolution to bind the glutamate dehydrogenase. The wells were then washedthoroughly to remove unbound horseradish peroxidase. One hundredmicroliters of substrate solution was then added to each well, incubated5 minutes, and then the reaction was stopped by adding 50 microliters ofdilute acid solution. Results were read at 450 nm on an ELISA reader.Positive samples had an optical density greater than 0.12.

Table 13 shows the results of the assays, and compares the results ofthe method and device of the present invention with the ELISA assay.

TABLE 13 Comparison of the method and device of the invention with anELISA Method N = 49 Ag Invention Pos Ag Invention Neg C. diff. Chek pos9  0 C. diff. Chek neg 0 40 Sensitivity 100.0 Specificity 100.0Predicted Positive Value 100.0 Predicted Negative Value 100.0Correlation 100.0

The results indicate that the device and method of the present inventionproduce results that are identical to the sensitive ELISA method used.Thus, the device and method of the invention are suitable for detectionof numerous substances of interest.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the practice of the presentmethod and in construction and use of the present device withoutdeparting from the scope or spirit of the invention. Other embodimentsof the invention will be apparent to those skilled in the art fromconsideration of the specification and practice of the invention. It isintended that the specification and examples be considered as exemplaryonly.

The invention claimed is:
 1. A device for detecting the presence oramount of a substance of interest in a liquid sample, the devicecomprising: a sample receiving zone for receiving the liquid sample,wherein the sample receiving zone is present on a porous material; asample filtering zone for filtering the liquid sample received at thesample receiving zone, wherein the sample filtering zone is present onthe porous material, and wherein the sample receiving zone and thesample filtering zone are integral with the porous material; a porousmembrane comprising a specific binding pair member at a detection zonethat specifically binds to the substance of interest or a substancebound to the substance of interest, wherein the porous membrane is aseparate element from the porous material, and wherein the porousmaterial and porous membrane are in physical contact over at least anarea comprising a portion of the detection zone, and wherein the porousmaterial and porous membrane are in physical contact in a configurationthat permits liquid present in the liquid sample to diffuse in, out,through, and about the porous membrane in a substantially random,non-directional manner in an area comprising at least a portion of thedetection zone.
 2. The device of claim 1, further comprising a containerthat contains at least a portion of the porous material and porousmembrane.
 3. The device of claim 2, further comprising a porous materialcomprising a wash solution receiving zone.
 4. The device of claim 1,wherein the specific binding pair member comprises a biological bindingpair member.
 5. The device of claim 4, wherein the biological bindingpair member comprises a protein, a nucleic acid, biotin, avidin,streptavidin, or combination thereof.
 6. The device of claim 4, whereinthe biological binding pair member comprises one or more proteins. 7.The device of claim 6, wherein the one or more proteins comprises anantibody.